Methods of treating lung cancer by administration of an alk inhibitor

ABSTRACT

Novel gene deletions and translocations involving chromosome 2 resulting in fusion proteins combining part of Anaplastic Lymphoma Kinase (ALK) kinase with part of a secondary protein have now been identified in human solid tumors, e.g. non-small cell lung carcinoma (NSCLC). Secondary proteins include Echinoderm Microtubule-Associated Protein-Like 4 (EML-4) and TRK-Fusion Gene (TFG). The EML4-ALK fusion protein, which retains ALK tyrosine kinase activity, was confirmed to drive the proliferation and survival of NSCLC characterized by this mutation. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant polypeptides, probes for detecting it, isolated mutant polypeptides, and reagents for detecting the fusion and truncated polypeptides. The invention also provides methods for determining the presence of these mutant polypeptides in a biological sample, methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer characterized by the mutant polynucleotides or polypeptides.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/940,521 filed Jul. 12, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/438,218 filed Apr. 3, 2012, now U.S. Pat. No.8,486,645, which is a divisional of U.S. patent application Ser. No.12/589,176 filed Oct. 19, 2009, now U.S. Pat. No. 8,168,383, which is acontinuation-in-part of U.S. application Ser. No. 11/787,132 filed Apr.13, 2007, now U.S. Pat. No. 7,700,339, which claims priority to and thebenefit of, U.S. Ser. No. 60/792,364 filed Apr. 14, 2006, now expired,the disclosures of which are incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

The invention relates generally to proteins and genes involved incancer, and to the detection, diagnosis and treatment of cancer.

BACKGROUND OF THE INVENTION

Many cancers are characterized by disruptions in cellular signalingpathways that lead to aberrant control of cellular processes, or touncontrolled growth and proliferation of cells. These disruptions areoften caused by changes in the activity of particular signalingproteins, such as kinases. Among these cancers are solid tumors, likenon-small cell lung carcinoma (NSCLC). NSCLC is the leading cause ofcancer death in the United States, and accounts for about 87% of alllung cancers. There are about 151,000 new cases of NSCLC in the UnitedStates annually, and it is estimated that over 120,000 patients will dieannually from the disease in the United States alone. See “Cancer Factsand FIGS. 2005,” American Cancer Society. NSCLC, which comprises threedistinct subtypes, is often only detected after it has metastasized, andthus the mortality rate is 75% within two years of diagnosis.

It is known that gene deletions and/or translocations resulting inkinase fusion proteins with aberrant signaling activity can directlylead to certain cancers. For example, it has been directly demonstratedthat the BCR-ABL oncoprotein, a tyrosine kinase fusion protein, is thecausative agent in human chronic myelogenous leukemia (CML). The BCR-ABLoncoprotein, which is found in at least 90-95% of CML cases, isgenerated by the translocation of gene sequences from the c-ABL proteintyrosine kinase on chromosome 9 into BCR sequences on chromosome 22,producing the so-called Philadelphia chromosome. See, e.g. Kurzock etal., N. Engl. J. Med. 319: 990-998 (1988). The translocation is alsoobserved in acute lymphocytic leukemia and NSCLC cases.

Gene translocations and deletions leading to mutant or fusion proteinsimplicated in a variety of other cancers have been described. Forexample, Falini et al., Blood 99(2): 409-426 (2002), reviewtranslocations known to occur in hematological cancers, including theNPM-ALK fusion found in ALCL. To date, only a limited number of genetranslocations, deletions, and mutant proteins occurring in lung cancershave been described, including the t(15;19) translocation involvingNotch3. See Dang et al., J. Natl. Can. Instit. 92(16): 1355-1357 (2000).Defects in RNA Binding Protein-6 (EML-4) expression and/or activity havebeen found in small cell and non-small cell lung carcinomas. See Drabkinet al., Oncogene 8(16): 2589-97 (1999). However, to date, notranslocations or deletions in human NSCLC cancer that involve proteinkinases have been described.

Defects in ALK kinase expression resulting from the fusion of NPM to ALKin large cell anaplastic lymphoma have been described. See Morris etal., 1994; Shiota et al., 1994. The fusion of ALK to moesin, non-musclemyosin heavy chain 9 (Tort et al. 2001), clarthrin heavy chain (Touriolet al., 2000; Bridge et al., 2001), tropomyosin 3 (TPM3) (Lamant et al.,1999), TRK-fused gene (TGF) (Hernandez et al., Am. J. Path. 160(4):1487-1493 (2002)) and other genes have been described. In particular,the TGF-ALK fusion was reported in non-solid lymphoma, but to date thisfusion has not been described in solid tumors. The general role of ALKin cancer has been described. See Pulford et al., J. Cell Physiol.199(3): 330-358 (2004). However, to date, no defects in EML-4 expressionand/or activation have been described.

Identifying mutations in human cancers is highly desirable because itcan lead to the development of new therapeutics that target such fusionor mutant proteins, and to new diagnostics for identifying patients thathave such gene mutations. For example, BCR-ABL has become a target forthe development of therapeutics to treat leukemia. Most recently,Gleevec® (Imatinib mesylate, STI-571), a small molecule inhibitor of theABL kinase, has been approved for the treatment of CML. This drug is thefirst of a new class of anti-proliferative agents designed to interferewith the signaling pathways that drive the growth of tumor cells. Thedevelopment of this drug represents a significant advance over theconventional therapies for CML and ALL, chemotherapy and radiation,which are plagued by well known side-effects and are often of limitedeffect since they fail to specifically target the underlying causes ofthe malignancies. Likewise, reagents and methods for specificallydetecting BCR-ABL fusion protein in patients, in order to identifypatients most likely to respond to targeted inhibitors like Gleevec®,have been described.

Accordingly, there remains a need for the identification of novel genemutations, such as translocations or deletions, resulting in fusion ormutant proteins implicated in the progression of human cancers,particularly solid tumors, including lung cancers like NSCLC, and thedevelopment of new reagents and methods for the study and detection ofsuch fusion proteins. Identification of such fusion proteins will, amongother things, desirably enable new methods for selecting patients fortargeted therapies, as well as for the screening of new drugs thatinhibit such mutant/fusion proteins.

SUMMARY OF THE INVENTION

In accordance with the invention, novel gene deletion mutationsoccurring in human chromosome 2 that result in fusion proteins combiningpart of Anaplastic Lymphoma Kinase (ALK) with a secondary protein havenow been identified in the human solid tumor non-small cell lungcarcinoma (NSCLC). Secondary proteins involved in the ALK fusionsinclude Echinoderm Microtubule-Associated Protein-Like 4 (EML-4) andTRK-Fused Gene (TFG). The mutant/fusion ALK kinases have presently beenobserved in non-small cell lung carcinoma patient samples.

The invention therefore provides, in part, isolated polynucleotides andvectors encoding the disclosed mutant/fusion ALK polypeptides, probesand assays for detecting them, isolated mutant/fusion ALK polypeptides,recombinant mutant polypeptides, and reagents for detecting the mutantALK polynucleotides and polypeptides. The disclosed identification ofthese new mutant ALK kinases and translocations/deletions enables newmethods for determining the presence of mutant ALK polynucleotides orpolypeptides in a biological sample, methods for screening for compoundsthat inhibit the mutant kinase protein, and methods for inhibiting theprogression of a cancer characterized by the expression of mutant ALKpolynucleotides or polypeptides, which are also provided by theinvention.

Accordingly, in a first aspect, the invention provides an isolatedpolynucleotide comprising a nucleotide sequence at least 95% identicalto a sequence selected from the group consisting of: (a) a nucleotidesequence encoding an EML4-ALK fusion polypeptide comprising theN-terminal amino acid sequence of EML-4 (residues 1-222 of SEQ ID NO: 3)and the kinase domain of ALK (residues 1116-1383 of SEQ ID NO: 5); anucleotide sequence encoding a EML4-ALK fusion polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 18; (b) anucleotide sequence encoding a EML4-ALK fusion polypeptide, saidnucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2or SEQ ID NO: 19; (c) a nucleotide sequence comprising at least sixcontiguous nucleotides encompassing the fusion junction of a EML4-ALKfusion polynucleotide; (d) a nucleotide sequence complementary to any ofthe nucleotide sequences of (a)-(d).

In another aspect, the invention provides an isolated polynucleotidethat hybridizes under stringent hybridization conditions to apolynucleotide of the first aspect. In some embodiments, said isolatedpolynucleotide that hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.

In some embodiments, the polynucleotide further comprises a detectablelabel.

In another aspect, the invention provides a method for producing arecombinant vector by inserting a polynucleotide of the invention into avector, and provides the recombinant vector so produced. In someembodiments, the recombinant vector of the invention is an expressionvector.

In additional aspects, the invention provides a method for making arecombinant host cell comprising introducing the recombinant vector ofthe invention into a host cell, and the recombinant host cell soproduced.

In yet another aspect, the invention provides a method for producing arecombinant EML4-ALK fusion polypeptide comprising culturing therecombinant host cell of the invention under conditions suitable for theexpression of said fusion polypeptide and recovering said polypeptide.

In a further aspect, the invention provides an isolated polypeptidecomprising an amino acid sequence at least 95% identical to a sequenceselected from the group consisting of: (a) an amino acid sequenceencoding a EML4-ALK fusion polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 18; (b) an amino acid sequenceencoding a EML4-ALK fusion polypeptide comprising the N-terminal aminoacid sequence of EML4 (1-222 of SEQ ID NO: 3 and the kinase domain ofALK (residues 1116-1383 of SEQ ID NO: 5); and (c) an amino acid sequenceencoding a polypeptide comprising at least six contiguous amino acidsencompassing the fusion junction (residues 233-234 of SEQ ID NO: 1 orresidues 495-496 of SEQ ID NO: 18) of a EML4-ALK fusion polypeptide.

In further embodiments, the invention provides a recombinant EML4-ALKfusion polypeptide or truncated ALK kinase polypeptide produced usingthe recombinant vector or the recombinant host cell of the invention.

In a further aspect, the invention provides an isolated reagent thatspecifically binds to or detects a EML4-ALK fusion polypeptide of theinvention but does not bind to or detect either wild type EML4 or wildtype ALK. In some embodiments, the reagent is an antibody or aheavy-isotope labeled (AQUA) peptide. In some embodiments, the isotopelabeled (AQUA) peptide comprises the amino acid sequence of the fusionjunction of EML4-ALK fusion polypeptide.

In another aspect, the invention provides a method for detecting thepresence of a mutant ALK polynucleotide and/or polypeptide in a cancer,said method comprising the steps of: (a) obtaining a biological samplefrom a patient having or suspected of having cancer; and (b) utilizingat least one reagent that specifically binds to or detectspolynucleotide and/or a polypeptide of the invention (e.g., an EML4-ALKfusion polynucleotide or a EML4-ALK fusion polypeptide) to determinewhether a EML4-ALK fusion polynucleotide and/or polypeptide is presentin said biological sample. In some embodiments, the biological samplecomprises a circulating tumor cell.

In another aspect, the invention provides a method for identifying apatient comprising a cancer comprising a mutant ALK polynucleotideand/or polypeptide, comprising: (a) obtaining a circulating tumor cellfrom a patient having or suspected of having cancer; and (b) utilizingat least one reagent that specifically binds to a EML4-ALKpolynucleotide and/or at least one reagent that specifically binds to aEML4-ALK polypeptide to determine whether a EML4-ALK fusionpolynucleotide and/or polypeptide is present in said circulating tumorcell, wherein the specific binding of said reagent to saidpolynucleotide or detection of said polypeptide by said reagentidentifies said patient as comprising a cancer comprising a mutant ALKpolynucleotide and/or polypeptide.

In some embodiments, the cancer is lung cancer (e.g., non-small celllung carcinoma (NSCLC)). In some embodiments, the presence of a mutantEML4-ALK polynucleotide or polypeptide identifies a cancer that islikely to respond to a composition comprising at least one ALKkinase-inhibiting therapeutic. In some embodiments, the method isimplemented in a flow-cytometry (FC), immuno-histochemistry (IHC), orimmuno-fluorescence (IF) assay format. In some embodiments, the methodis implemented in a fluorescence in situ hybridization (FISH) orpolymerase chain reaction (PCR) assay format. In some embodiments, themethod is implemented in at least two assay formats selected from thegroup consisting of flow-cytometry (FC), immuno-histochemistry (IHC),immuno-fluorescence (IF), fluorescence in situ hybridization (FISH) andpolymerase chain reaction (PCR).

In some embodiments, the activity of said EML4-ALK fusion polypeptide isdetected.

In a further aspect, the invention provides a method for determiningwhether a compound inhibits the progression of a cancer characterized bya EML4-ALK fusion polynucleotide and/or polypeptide, said methodcomprising the step of determining whether said compound inhibits theexpression and/or activity of said EML4-ALK fusion polypeptide in saidcancer.

In some embodiments, the inhibition of expression and/or activity ofsaid EML4-ALK fusion polypeptide is determined using at least onereagent that detects a polynucleotide of the invention and/or at leastone reagent that specifically binds to a polypeptide of the invention.

In a further aspect, the invention provides a method for inhibiting theprogression of a cancer that expresses an EML4-ALK fusion polypeptide,said method comprising the step of inhibiting the expression and/oractivity of said EML4-ALK fusion polypeptide in said cancer.

In some embodiments, the cancer is lung cancer (e.g., a non-small celllung carcinoma (NSCLC)).

In another aspect, the invention provides a kit for the detection of aEML4-ALK fusion polynucleotide and/or polypeptide in a biologicalsample, said kit comprising at least one polynucleotide of the inventionand/or at least one reagent that specifically binds to a polypeptide ofthe invention, and one or more secondary reagents.

The aspects and embodiments of the invention are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A—shows the locations of the EML-4 gene and ALK gene on chromosome2 (panel A), and the domain locations of full-length EML-4 and ALKproteins as well as those of EML4-ALK fusion protein (short variant)(panel B); the fusion junction occurs at amino acids 233-234, and thefusion protein includes the kinase domain (but not the transmembrane andextracellular domains) of ALK. Also shown (in panel B) the DNA (andprotein) sequence of the EML4 exon 6/intron6/ALK exon 20 fusion junctionregion (SEQ ID NO: 7 and SEQ ID NO: 8, respectively).

FIG. 1B—shows the locations of the EML-4 gene and ALK gene on chromosome2 (panel A), and the domain locations of full-length EML-4 and ALKproteins as well as those of EML4-ALK fusion protein (long variant)(panel B); the fusion junction occurs at amino acids 495-496, and thefusion protein includes the kinase domain (but not the transmembrane andextracellular domains) of ALK. Also shown (in panel B) the DNA (andprotein) sequence of the EML4 exon 13/ALK exon 20 fusion junction region(SEQ ID NO: 24 and SEQ ID NO: 25, respectively).

FIG. 1C—shows the locations of the TFG gene on chromosome 6 and ALK geneon chromosome 2 (panel A), and the domain locations of full-length TFGand ALK proteins as well as those of TFG-ALK fusion protein (panel B);the fusion junction occurs at amino acids 138-139, and the fusionprotein includes the kinase domain (but not the transmembrane andextracellular domains) of ALK. Also shown (in panel B) the DNA (andprotein) sequence of the TFG exon 3/ALK exon 20 fusion junction region(SEQ ID NO: 26 and SEQ ID NO: 27, respectively).

FIG. 2A—is the amino acid sequence (1 letter code) of human EML4-ALKfusion protein (short variant) (SEQ ID NO: 1) (top panel) with codingDNA sequence also indicated (SEQ ID NO: 2) (bottom panel); the residuesof the EML-4 moiety are in italics, while the residues of the kinasedomain of ALK are in bold.

FIG. 2B—is the amino acid sequence (1 letter code) of human EML4-ALKfusion protein (long variant) (SEQ ID NO: 18) (top panel) with codingDNA sequence also indicated (SEQ ID NO: 19) (bottom panel); the residuesof the EML-4 moiety are in italics, while the residues of the kinasedomain of ALK are in bold.

FIG. 2C—is the amino acid sequence (1 letter code) of human TFG-ALKfusion protein (SEQ ID NO: 20) (top panel) with coding DNA sequence alsoindicated (SEQ ID NO: 21) (bottom panel); the residues of the TFG moietyare in italics, while the residues of the kinase domain of ALK are inbold.

FIG. 3A-3B—is the amino acid sequence (1 letter code) of human EML-4protein (SEQ ID NO: 3) (SwissProt Accession No. 061936) with coding DNAsequence also indicated (SEQ ID NO:4) (GeneBank Accession No. NM019063);the residues retained in the short variant deletion mutant areunderlined, while the residues retained in long variant and italicized.

FIG. 4A-4B—is the amino acid sequence (1 letter code) of human ALKkinase (SEQ ID NO: 5) (SwissProt Accession No. Q9UM73) with coding DNAsequence also indicated (SEQ ID NO: 6) (GeneBank Accession No.HSU66559); the residues retained in the deletion mutants are underlined,while the residues of the kinase domain are in bold.

FIG. 4C-4D—is the amino acid sequence (1 letter code) of human TFGprotein (SEQ ID NO: 22) (SwissProt Accession No. Q92734) with coding DNAsequence also indicated (SEQ ID NO: 23) (GeneBank Accession No.NM006070); the residues retained in the deletion mutant are underlined.

FIGS. 5A-D—are images of gels depicting (A) detection of ALK via the 5′RACE product with ALK primers after 2 rounds of PCR; UAP stands forUniversal Amplification Primer, GSP for Gene Specific Primer, (B)detection of the fusion gene formed by the EML-4 and ALK deletion mutantby RT-PCR, (C) detection of the EML4-ALK fusion gene (short and longvariants) in human NSCLC tumor samples by 5′ RACE, and (D) detection ofthe TFG-ALK fusion gene in human NSCLC tumor samples by 5′ RACE.

FIG. 6—is an image depicting the detection of the fusion gene formed bythe EML-4 and ALK translocation in H2228 cells by FISH assay employing adual-color (orange/green) break-apart probe comprising probes toopposite sides of the ALK gene breakpoint 2p23; probe sizes andlocations are shown in the upper panel.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, previously unknown gene deletions andtranslocations that result in mutant kinase fusion proteins, combiningpart of Anaplastic Lymphoma Kinase (ALK) with a portion of a secondaryprotein, have now been identified in the human solid tumor non-smallcell lung carcinoma (NSCLC). Secondary proteins involved in thediscovered ALK fusions include Echinoderm Microtubule-AssociatedProtein-Like 4 (EML-4) and TRK-Fused Gene (TFG).

The two disclosed deletions, which occurs between the EML4 and ALK geneson chromosome 2, produce fusion proteins that combines the N-terminus ofEML-4, a 401 amino acid microtubule binding protein, with the kinasedomain and c-terminus of ALK, a 1620 amino acid membrane tyrosinekinase. The resulting EML4-ALK fusion proteins, which are 796 aminoacids (short variant) and 1059 amino acids (long variant) respectively,and retain ALK kinase activity, are expected to drive the proliferationand survival of a subset of human solid tumors, including NSCLC.

The disclosed translocation, which occurs between the TFG gene onchromosome 6 and the ALK gene on chromosome 2, produces a fusion proteinthat combines the N-terminus of TFG, a 400 amino acid protein, with thekinase domain and c-terminus of ALK, a 1620 amino acid membrane tyrosinekinase. The resulting TFG-ALK fusion protein, which is 701 amino acids,has previously been observed in non-solid human lymphoma (Hernandez etal. (2002), supra.), but has not previously been described in solidtumors. The TFG-ALK fusion protein retains ALK kinase activity, and isexpected to drive the proliferation and survival of a subset of humansolid tumors, including NSCLC.

Although a few gene translocations or deletions that result in aberrantfusion proteins have been described in NSCLC, including the t(15;19)translocation involving Notch3 (see Dang et al., supra.), the presentlydisclosed EML4-ALK deletion mutants and fusion protein are novel.Similarly, the TFG-ALK translocation mutant and fusion protein, thoughknown in non-solid tumors like lymphoma, is novel in the solid tumorNSCLC. EML-4 is a microtubule-associated protein that is expressed inmost human tissues. To date, no defects in EML-4 expression and/oractivity have been reported. ALK is a membrane tyrosine kinase, and isexpressed, in humans, in brain and CNS tissues, also small intestine andtestis, but not in normal lymphoid cells. It plays an important role inthe normal development and function of the nervous system (Iwahara etal., 1997).

Defects in ALK expression and/or activation have been found in largecell anaplastic lymphoma and neuroblastoma (see Morris et al., 1994,Osajima-Hakomori et al., 2005). The fusion of ALK to moesin, non-musclemyosin heavy chain 9, clarthrin heavy chain, tropomyosin 3 (TPM3),TRK-fused gene (TFG), and other genes has been described. See Tort etal.; Touriol et al., Hernandez et al., supra.). Interestingly, thedisclosed fusion of EML-4 to ALK (short variant) occurs at precisely thesame point in wild type ALK (amino acid 1058) as previously describedfor other ALK fusion mutants.

As further described below, the EML4-ALK deletion mutants and theexpressed fusion proteins have presently been isolated and sequenced,and cDNAs for expressing the fusion proteins produced. Accordingly, theinvention provides, in part, isolated polynucleotides that encodeEML4-ALK fusion polypeptides, nucleic acid probes that hybridize to suchpolynucleotides, and methods, vectors, and host cells for utilizing suchpolynucleotides to produce recombinant mutant ALK polypeptides. Theinvention also provides, in part, isolated polypeptides comprising aminoacid sequences encoding EML4-ALK fusion polypeptides, recombinant mutantpolypeptides, and isolated reagents that specifically bind to and/ordetect EML4-ALK fusion polypeptides, but do not bind to or detect eitherwild type EML-4 or wild type ALK. These aspects of the invention, whichare described in further detail below, will be useful, inter alia, infurther studying the mechanisms of cancers driven by mutant ALK kinaseexpression/activity, for identifying solid tumors (e.g. carcinomasincluding lung carcinomas and sarcomas) and other cancers characterizedby the disclosed ALK deletion and translocation mutations and/or fusionprotein, or expression/activity of mutant ALK kinase, and in practicingmethods of the invention as further described below.

The identification of the novel ALK kinase mutants and gene deletion andtranslocation mutations has important implications for the potentialdiagnosis and treatment of solid tumors, such as NSCLC, that arecharacterized by one or more of these fusion proteins. NSCLC, forexample, is often only detected after it has metastasized, and thus themortality rate is 75% within two years of diagnosis. Accordingly, theability to identify, as early as possible, patients having genemutations that may lead to NSCLC, would be highly desirable.

Therefore, the discovery of the EML4-ALK fusion proteins (short and longvariants) resulting from gene deletion and the TFG-ALK fusion proteinresulting from gene translocation, which are expected to driveproliferation and survival of a solid tumor, NSCLC, enables importantnew methods for accurately identifying mammalian solid tumors, includinglung cancers (such as NSCLC), as well as other cancers, in which an ALKfusion protein (such as EML4-ALK or TFG-ALK) is expressed. These tumorsare most likely to respond to inhibitors of the kinase activity of themutant ALK protein, such as WHI-131 or WHI-154. The ability to identify,as early as possible, cancers that are driven by a mutant ALK kinasewill greatly assist in clinically determining which therapeutic, orcombination of therapeutics, will be most appropriate for a particularpatient, thus helping to avoid prescription of inhibitors targetingother kinases that are not, in fact, the primary signaling moleculedriving the cancer.

Accordingly, the invention provides, in part, methods for detecting thepresence of an ALK mutant polynucleotide and/or fusion polypeptide in acancer using fusion-specific and mutant-specific reagents of theinvention. Such methods may be practiced, for example, to identify asolid tumor, such as NSCLC, that is likely to respond to an inhibitor ofthe ALK kinase activity of the mutant protein. The invention alsoprovides, in part, methods for determining whether a compound inhibitsthe progression of a cancer characterized by an EML4-ALK fusionpolypeptide. Further provided by the invention is a method forinhibiting the progression of a solid tumor that expresses an EML4-ALKfusion polypeptide or a TFG-ALK fusion polypeptide by inhibiting theexpression and/or activity of the mutant polypeptide. Such methods aredescribed in further detail below.

The further aspects, advantages, and embodiments of the invention aredescribed in more detail below. All references cited herein are herebyincorporated by reference in their entirety.

Definitions

As used herein, the following terms have the meanings indicated.

“Antibody” or “antibodies” refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE, including Ft orantigen-recognition fragments thereof, including chimeric, polyclonal,and monoclonal antibodies. The term “humanized antibody”, as usedherein, refers to antibody molecules in which amino acids have beenreplaced in the non-antigen binding regions in order to more closelyresemble a human antibody, while still retaining the original bindingability.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic EML4-ALK or TFG-ALK fusionpolypeptide, or any oligopeptide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

The term “biological sample” is used in its broadest sense, and meansany biological sample suspected of containing ALK fusion polynucleotidesor polypeptides or fragments thereof (including EML4-ALK and TFG-ALKfusion polynucleotides and polypeptides), and may comprise a cell,chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), genomic DNA (in solution or bound to a solid support suchas for Southern analysis), RNA (in solution or bound to a solid supportsuch as for northern analysis), cDNA (in solution or bound to a solidsupport), an extract from cells, blood, urine, marrow, or a tissue, andthe like.

“Characterized by” with respect to a cancer and mutant ALKpolynucleotide and polypeptide is meant a cancer in which a genedeletion or translocation and/or expressed fusion polypeptide involvingALK are present as compared to a cancer in which such gene deletionand/or fusion polypeptide are not present. The presence of mutantpolypeptide may drive, in whole or in part, the growth and survival ofsuch cancer.

“Consensus” refers to a nucleic acid sequence which has beenre-sequenced to resolve uncalled bases, or which has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′ directionand re-sequenced, or which has been assembled from the overlappingsequences of more than one Incyte clone using the GELVIEW™ FragmentAssembly system (GCG, Madison, Wis.), or which has been both extendedand assembled.

“ALK kinase-inhibiting therapeutic” means any composition comprising oneor more compounds, chemical or biological, which inhibits, eitherdirectly or indirectly, the expression and/or activity of wild type ortruncated ALK kinase, either alone and/or as part of a fusion protein(such as EML4-ALK fusion proteins and TFG-ALK fusion protein).

“Derivative” refers to the chemical modification of a nucleic acidsequence encoding a disclosed fusion polynucleotide or the encodedpolypeptide itself. Illustrative of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative would encode a polypeptide that retains essentialbiological characteristics of the natural molecule.

“Detectable label” with respect to a polypeptide, polynucleotide, orreagent disclosed herein means a chemical, biological, or othermodification, including but not limited to fluorescence, mass, residue,dye, radioisotope, label, or tag modifications, etc., by which thepresence of the molecule of interest may be detected.

“Expression” or “expressed” with respect to an ALK fusion polypeptide ina biological sample means significantly expressed as compared to controlsample in which this fusion polypeptide is not significantly expressed.

“Heavy-isotope labeled peptide” (used interchangeably with AQUA peptide)means a peptide comprising at least one heavy-isotope label, which issuitable for absolute quantification or detection of a protein asdescribed in WO/03016861, “Absolute Quantification of Proteins andModified Forms Thereof by Multistage Mass Spectrometry” (Gygi et al.),further discussed below. The term “specifically detects” with respect tosuch an AQUA peptide means the peptide will only detect and quantifypolypeptides and proteins that contain the AQUA peptide sequence andwill not substantially detect polypeptides and proteins that do notcontain the AQUA peptide sequence.

“Isolated” (or “substantially purified”) refers to nucleic or amino acidsequences that are removed from their natural environment, isolated orseparated. They preferably are at least 60% free, more preferably 75%free, and most preferably 90% or more free from other components withwhich they are naturally associated.

“Mimetic” refers to a molecule, the structure of which is developed fromknowledge of the structure of an ALK fusion polypeptide or portionsthereof and, as such, is able to effect some or all of the actions oftranslocation associated protein-like molecules.

“Mutant ALK” or “fusion” polynucleotide or polypeptide means a fusionpolynucleotide or polypeptide involving ALK and a secondary protein(e.g. EML-4 or TFG), as described herein.

“Polynucleotide” (or “nucleotide sequence”) refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin,which may be single- or double-stranded, and represent the sense oranti-sense strand.

“Polypeptide” (or “amino acid sequence”) refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms, such as “polypeptide” or “protein”, are not meant to limit theamino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule.

“EML4-ALK fusion polynucleotide” refers to the nucleic acid sequence ofa substantially purified EML4-ALK deletion mutant gene product or fusionpolynucleotide (short or long variant) as described herein, obtainedfrom any species, particularly mammalian, including bovine, ovine,porcine, murine, equine, and preferably human, from any source whethernatural, synthetic, semi-synthetic, or recombinant.

“EML4-ALK fusion polypeptide” refers to the amino acid sequence of asubstantially purified EML4-ALK fusion polypeptide (short or longvariant) described herein, obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

“TFG-ALK fusion polynucleotide” refers to the nucleic acid sequence of asubstantially purified TFG-ALK translocation mutant gene product orfusion polynucleotide as described herein, obtained from any species,particularly mammalian, including bovine, ovine, porcine, murine,equine, and preferably human, from any source whether natural,synthetic, semi-synthetic, or recombinant.

“TFG-ALK fusion polypeptide” refers to the amino acid sequence of asubstantially purified TFG-ALK fusion polypeptide described herein,obtained from any species, particularly mammalian, including bovine,ovine, porcine, murine, equine, and preferably human, from any sourcewhether natural, synthetic, semi-synthetic, or recombinant.

The terms “specifically binds to” (or “specifically binding” or“specific binding”) in reference to the interaction of an antibody and aprotein or peptide, mean that the interaction is dependent upon thepresence of a particular structure (i.e. the antigenic determinant orepitope) on the protein; in other words, the antibody is recognizing andbinding to a specific protein structure rather than to proteins ingeneral. The term “does not bind” with respect to an antibody's bindingto sequences or antigenic determinants other than that for which it isspecific means does not substantially react with as compared to theantibody's binding to antigenic determinant or sequence for which theantibody is specific.

The term “stringent conditions” with respect to sequence or probehybridization conditions is the “stringency” that occurs within a rangefrom about T_(m) minus 5° C. (5° C. below the melting temperature(T_(m)) of the probe or sequence) to about 20° C. to 25° C. below T_(m).Typical stringent conditions are: overnight incubation at 42° C. in asolution comprising: 50% formamide, 5×.SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 micrograms/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65° C. As willbe understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

A “variant” of a mutant ALK polypeptide refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A. Identification of Mutant ALK Kinases in Human Solid Tumors.

The novel human gene deletions disclosed herein, which occurs onchromosome 2 and result in expression of two fusion protein variantsthat combine the N-terminus of EML-4 with the kinase domain andC-terminus of ALK, were surprisingly identified during examination ofglobal phosphorylated peptide profiles in extracts from non-small celllung carcinoma (NSCLC) cell lines (including H2228) and solid tumorsfrom patients. NSCLC, a solid tumor, is a subtype of lung cancer. Theproteins involved in these deletion fusions are shown in FIGS. 1A-1B,panel A.

The phosphorylation profile of the H2228 cell line was first elucidatedusing a recently described technique for the isolation and massspectrometric characterization of modified peptides from complexmixtures (see U.S. Patent Publication No. 20030044848, Rush et al.,“Immunoaffinity Isolation of Modified Peptides from Complex Mixtures”(the “IAP” technique), as further described in Example 1 below.Application of the IAP technique using a phosphotyrosine-specificantibody (CELL SIGNALING TECHNOLOGY, INC., Beverly, Mass., 2003/04 Cat.#9411), identified that the H2228 cell line expresses ALK kinase, butthat the protein was apparently truncated. The screen identified manyother activated kinases in the cell line, including some that are knownto be activated in lung cancer. Analysis of the sequence 5′ to ALK by 5′RACE then identified that the kinase was fused to the N-terminus ofEML-4 (see FIG. 6).

Subsequent examination of 154 tumor samples from NSCLC patients usingthe same global phospho-profiling approach not only confirmed thepresence of the EML4-ALK (short variant) mutation in a population ofthose patients, but also revealed the presence of a second EML4-ALK(long variant) and the presence of the TFG-ALK mutation in other patientpopulations (see Example 1B and 1C).

Confirmation that the mutant ALK proteins are driving cell proliferationand survival in these NSCLC tumors may be established by inhibiting thecells using siRNA silencing (see Example 3).

The EML4-ALK fusion genes (short and long variants) and the TFG-ALKfusion gene were amplified by PCR, isolated, and sequenced (see Example3). As shown in panel B of FIGS. 1A-1B, the EML4-ALK deletion combinesthe N-terminus of wild type EML-4 (either amino acids 1-233 in the shortvariant, or amino acids 1-495 in the long variant) with the kinasedomain and C-terminus of wild type ALK (amino acids 1057-1620) (see alsoSEQ ID NOs: 3 and 5). The fusion junction occurs just C-terminus to thetransmembrane domain of wild type ALK (see FIGS. 1A-1B). The EML4-ALKfusion polypeptides retain the N-terminal 233 or 495 amino acids ofEML-4, respectively, which includes the coiled coil domain of thisprotein. The resulting EML4-ALK fusion proteins, which comprise 796amino acids (short variant) or 1059 amino acids (long variant),respectively (see panel B of FIGS. 1A-1B and FIGS. 2A-2B (SEQ ID NOs: 1and 18)), retain kinase activity of ALK. The exons involved and thefusion junction are shown in FIGS. 1A-1B (panel B). The fusion junctionincludes intron 6 from EML-4, which follows exon 6 (short variant) orexon 13 from EML-4 (long variant).

As shown in panel B of FIG. 1C, the TFG-ALK translocation combines theN-terminus of wild type TFG (amino acids 1-138) with the kinase domainand C-terminus of wild type ALK (amino acids 1057-1620) (see also SEQ IDNOs: 22 and 5; and panel B of FIG. 1C and FIG. 4C (SEQ ID NOs: 20 and1)). The fusion junction occurs just C-terminus to the transmembranedomain of wild type ALK (see FIG. 1C) and retains kinase activity ofALK. The exons involved and the fusion junction are shown in FIG. 1C(panel B). The fusion junction includes exon 3 from TFG and exon 20 fromALK.

FISH probes were used to detect the presence of the EML4-ALK (shortvariant) fusion protein in a group of 400 paraffin-embedded human NSCLCtumor samples (see Examples 6 and 7; FIG. 6). The incidence of thisshort variant mutation in this sample size was very low. However,expression of the EML4-ALK fusion proteins (both short and longvariants), as well as the TFG-ALK fusion protein, was detected in higherincidence using the IAP technique to examine global phosphorylationprofiles in another group of 154 frozen human NSCLC tumor samples frompatients (see Example 1B).

B. Isolated Polynucleotides.

The present invention provides, in part, isolated polynucleotides thatencode EML4-ALK fusion polypeptides, nucleotide probes that hybridize tosuch polynucleotides, and methods, vectors, and host cells for utilizingsuch polynucleotides to produce recombinant fusion polypeptides.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were determined using an automated peptide sequencer (see Example2). As is known in the art for any DNA sequence determined by thisautomated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 90% identical, more typically at least about95% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence will be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each nucleotide sequence set forth herein ispresented as a sequence of deoxyribonucleotides (abbreviated A, G, C andT). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO: 2 set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of SEQ ID NO: 2 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

In one embodiment, the invention provides an isolated polynucleotidecomprising a nucleotide sequence at least 95% identical to a sequenceselected from the group consisting of:

(a) a nucleotide sequence encoding an Echinoderm Microtubule-AssociatedProtein-Like 4/Anaplastic Lymphoma Kinase (EML4-ALK) fusion polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18;

(b) a nucleotide sequence encoding an EML4-ALK fusion polypeptide, saidnucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2or SEQ ID NO: 19;

(c) a nucleotide sequence encoding an EML4-ALK fusion polypeptidecomprising the N-terminal amino acid sequence of EML-4 (residues 1-222of SEQ ID NO: 3 or residues 1-495 of SEQ ID NO: 3) and the kinase domainof ALK (residues 1116-1383 of SEQ ID NO: 5);

(d) a nucleotide sequence comprising the N-terminal nucleotide sequenceof EML-4 (nucleotides 1-700 of SEQ ID NO: 4 or nucleotides 1-1486 of SEQID NO: 4) and the kinase domain nucleotide sequence of ALK (nucleotides3348-4149 of SEQ ID NO: 6);

(e) a nucleotide sequence comprising at least six contiguous nucleotidesencompassing the fusion junction (nucleotides 700-701 of SEQ ID NO: 2 ornucleotides 1486-1487 of SEQ ID NO: 19) of an EML4-ALK fusionpolynucleotide;

(f) a nucleotide sequence encoding a polypeptide comprising at least sixcontiguous amino acids encompassing the fusion junction (residues233-234 of SEQ ID NO: 1 or residues 495-496 of SEQ ID NO: 18) of anEML4-ALK fusion polypeptide; and

(g) a nucleotide sequence complementary to any of the nucleotidesequences of (a)-(f).

Using the information provided herein, such as the nucleotide sequencein FIG. 2 (SEQ ID NO: 2), a nucleic acid molecule of the presentinvention encoding a mutant ALK polypeptide of the invention may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material. Illustrative of theinvention, the EML4-ALK fusion polynucleotide (short variant) describedin FIG. 2 (SEQ ID NO: 2) was isolated from genomic DNA from a humanNSCLC cell line (as further described in Example 2 below). The fusiongene can also be identified in genomic DNA or cDNA libraries in othercancers, including solid tumors, in which a disclosed EML4-ALK genedeletion (chromosome 2) occurs.

The determined nucleotide sequences of the EML4-ALK fusion genes (SEQ IDNOs: 2 and 19) encode kinase fusion proteins of 796 amino acids (shortvariant) and 1059 amino acids (long variant), respectively (see FIGS.2A-B (SEQ ID NOs: 1 and 18) and FIGS. 1A-B). The EML4-ALK fusionpolynucleotides comprise the portion of the nucleotide sequence of wildtype EML-4 (see FIG. 3 (SEQ ID NO: 4)) that encodes the N-terminus(amino acids 1-233 (short variant) or amino acids 1-495 (long variant))of that protein with the portion of the nucleotide sequence of wild typeALK (see FIG. 4 (SEQ ID NO: 6)) that encodes the kinase domain andC-terminus of that protein. See FIGS. 1A-B. The kinase domain comprisesresidues 292-568 in the short variant fusion protein (encoded bynucleotides 874-1704 of the short variant fusion polynucleotide) orresidues 555-831 in the long variant fusion protein (encoded bynucleotides 1663-2494 of the long variant fusion polynucleotide). SeeFIGS. 2A-2B.

As indicated, the present invention provides, in part, the mature formof the EML4-ALK fusion proteins. According to the signal hypothesis,proteins secreted by mammalian cells have a signal or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Most mammalian cells and even insect cells cleave secretedproteins with the same specificity. However, in some cases, cleavage ofa secreted protein is not entirely uniform, which results in two or moremature species on the protein. Further, it has long been known that thecleavage specificity of a secreted protein is ultimately determined bythe primary structure of the complete protein, that is, it is inherentin the amino acid sequence of the polypeptide.

By the mature EML4-ALK polypeptide having the amino acid sequenceencoded, e.g. by the deposited cDNA clone, is meant the mature form ofthis fusion protein produced by expression in a mammalian cell (e.g.,3T3 cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the deposited clone or other cloneencoding mature fusion polypeptide.

As indicated, polynucleotides of the present invention may be in theform of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

Isolated polynucleotides of the invention are nucleic acid molecules,DNA or RNA, which have been removed from their native environment. Forexample, recombinant DNA molecules contained in a vector are consideredisolated for the purposes of the present invention. Further examples ofisolated DNA molecules include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules include in vivo or invitro RNA transcripts of the DNA molecules of the present invention.Isolated nucleic acid molecules according to the present inventionfurther include such molecules produced synthetically.

Isolated polynucleotides of the invention include the DNA molecule shownin FIGS. 2A-B (SEQ ID NOs: 2 and 19), DNA molecules comprising thecoding sequence for the mature EML4-ALK fusion proteins shown in FIGS.1A-B (SEQ ID NOs: 1 and 18), and DNA molecules that comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode a ALK mutant polypeptide ofthe invention. The genetic code is well known in the art, thus, it wouldbe routine for one skilled in the art to generate such degeneratevariants.

In another embodiment, the invention provides an isolated polynucleotideencoding the EML4-ALK fusion polypeptide comprising the EML4-ALK fusionnucleotide sequence contained in the above-described deposited cDNAclone. Preferably, such nucleic acid molecule will encode the maturefusion polypeptide encoded by the deposited cDNA clone or another cloneexpressing a full length EML4-ALK fusion protein described herein. Inanother embodiment, the invention provides an isolated nucleotidesequence encoding an EML4-ALK fusion polypeptide comprising theN-terminal amino acid sequence of EML-4 (residues 1-222 of SEQ ID NO: 3or residues 1-495 of SEQ ID NO: 3) and the kinase domain of ALK(residues 1116-1383 of SEQ ID NO: 5). In one embodiment, the polypeptidecomprising the kinase domain of ALK comprises residues 1057-1620 of SEQID NO: 5 (see FIG. 1, panel B). In another embodiment, theaforementioned N-terminal amino acid sequence of EML-4 and kinase domainof ALK are encoded by nucleotide sequences comprising nucleotides 1-666of SEQ ID NO: 4 or nucleotides 1-1486 of SEQ ID NO: 4 and nucleotides3171-4860 of SEQ ID NO: 6, respectively.

The invention further provides isolated polynucleotides comprisingnucleotide sequences having a sequence complementary to one of themutant ALK polynucleotides of the invention. Such isolated molecules,particularly DNA molecules, are useful as probes for gene mapping, by insitu hybridization with chromosomes, and for detecting expression of aEML4-ALK fusion protein in human tissue, for instance, by Northern blotanalysis, as further described in Section F below.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedEML4-ALK polynucleotide of the invention is intended fragments at leastabout 15 nucleotides, and more preferably at least about 20 nucleotides,still more preferably at least about 30 nucleotides, and even morepreferably, at least about 40 nucleotides in length, which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments of about 50-1500 nucleotides in length are also usefulaccording to the present invention, as are fragments corresponding tomost, if not all, of the mutant ALK nucleotide sequences of thedeposited cDNAs or as shown in FIG. 2 (SEQ ID NO: 2) or other cloneexpressing the a fusion polynucleotide as shown in FIGS. 2A-B (SEQ IDNOs: 2 or 19). By a fragment at least 20 nucleotides in length, forexample, is intended fragments that include 20 or more contiguous basesfrom the respective nucleotide sequences from which the fragments arederived. Generation of such DNA fragments is routine to the skilledartisan, and may be accomplished, by way of example, by restrictionendonuclease cleavage or shearing by sonication of DNA obtainable fromthe deposited cDNA clone or synthesized according to the sequencedisclosed herein. Alternatively, such fragments can be directlygenerated synthetically.

Preferred nucleic acid fragments or probes of the present inventioninclude nucleic acid molecules encoding the fusion junction of theEML4-ALK fusion gene products (see FIGS. 1A-B, panel B). For example, incertain preferred embodiments, an isolated polynucleotide of theinvention comprises a nucleotide sequence/fragment comprising at leastsix contiguous nucleotides encompassing the fusion junction (nucleotides700-701 of SEQ ID NO: 2 or nucleotides 1486-1487 of SEQ ID NO: 19) of anEML4-ALK fusion polynucleotide (see FIGS. 1A-B, panel B (SEQ ID NOs: 8and 25)). In another preferred embodiment, an isolated polynucleotide ofthe invention comprises a nucleotide sequence/fragment that encodes apolypeptide comprising at least six contiguous amino acids encompassingthe fusion junction (residues 233-234 of SEQ ID NO: 1 or residues495-496 of SEQ ID NO: 18) of an EML4-ALK fusion polypeptide (see FIGS.1A-B, bottom panel (SEQ ID NOs: 7 and 24)).

In another aspect, the invention provides an isolated polynucleotidethat hybridizes under stringent hybridization conditions to a portion ofa mutant ALK polynucleotide of the invention as described herein. By“stringent hybridization conditions” is intended overnight incubation at42° C. in a solution comprising: 50% formamide, 5×.SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1×SSC at about65° C.

By a polynucleotide that hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g. the mature EML4-ALK fusion polynucleotidedescribed in FIG. 2 (SEQ ID NO: 2)), for instance, a portion 50-750 ntin length, or even to the entire length of the reference polynucleotide,are also useful as probes according to the present invention, as arepolynucleotides corresponding to most, if not all, of the nucleotidesequences of the deposited cDNAs or the nucleotide sequences shown inFIGS. 2A-B (SEQ ID NOs: 2 or 19), or FIGS. 1A-B (panel B)) (SEQ ID NOs:7 and 24).

By a portion of a polynucleotide of “at least 20 nucleotides in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide. As indicated, suchportions are useful diagnostically either as a probe according toconventional DNA hybridization techniques or as primers foramplification of a target sequence by the polymerase chain reaction(PCR), as described, for instance, in MOLECULAR CLONING, A LABORATORYMANUAL, 2^(nd) Ed., Sambrook, J., Fritsch, E. F. and Maniatis, T., eds.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),the entire disclosure of which is hereby incorporated herein byreference. Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the EML4-ALK sequenceshown in FIG. 2 (SEQ ID NO: 2)) or to a complementary stretch of T (orU) resides, would not be included in a polynucleotide of the inventionused to hybridize to a portion of a nucleic acid of the invention, sincesuch a polynucleotide would hybridize to any nucleic acid moleculecontaining a poly (A) stretch or the complement thereof (e.g.,practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention, whichencode a mutant ALK polypeptide of the invention, may include but arenot limited to those encoding the amino acid sequence of the maturepolypeptide, by itself; the coding sequence for the mature polypeptideand additional sequences, such as those encoding the leader or secretorysequence, such as a pre-, or pro- or pre-pro-protein sequence; thecoding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37:767 (1984). As discussed below, other such fusion proteinsinclude a EML4-ALK fusion polypeptide itself fused to Fc at the N- orC-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of an EML4-ALK fusion polypeptide disclosed herein. Variantsmay occur naturally, such as a natural allelic variant. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism. See, e.g. GENES II, Lewin,B., ed., John Wiley & Sons, New York (1985). Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities (e.g. kinase activity) of the mutant ALKpolypeptides disclosed herein. Also especially preferred in this regardare conservative substitutions.

Further embodiments of the invention include isolated polynucleotidescomprising a nucleotide sequence at least 90% identical, and morepreferably at least 95%, 96%, 97%, 98% or 99% identical, to a mutant ALKpolynucleotide of the invention (for example, a nucleotide sequenceencoding the EML4-ALK fusion polypeptide having the complete amino acidsequence shown in FIG. 2 (SEQ ID NO: 1; or a nucleotide sequenceencoding the N-terminal of EML-4 and the kinase domain of ALK (see FIG.1, panel B; and FIGS. 3 and 4); or a nucleotide complementary to suchexemplary sequences).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a mutant ALKpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the mutantALK polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences shown in FIGS. 2A-B (SEQ ID NOs: 2 and 19) or tothe nucleotide sequence of the deposited cDNA clones described above canbe determined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711. Bestfit uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981),to find the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referenceEML4-ALK fusion polynucleotide sequence or truncated ALK polynucleotidesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

The present invention includes in its scope nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 2 (SEQ ID NO: 2), or to the nucleic acidsequences of the deposited cDNAs, irrespective of whether they encode apolypeptide having ALK kinase activity. This is because even where aparticular nucleic acid molecule does not encode a fusion polypeptidehaving ALK kinase activity, one of skill in the art would still know howto use the nucleic acid molecule, for instance, as a hybridization probeor a polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving kinase include, inter alia, (1) isolating the EML4-ALK deletiongene, or truncated ALK gene, or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theEML4-ALK deletion gene or truncated ALK gene, as described in Verma etal., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press,New York (1988); and Northern Blot analysis for detecting EML4-ALKfusion protein or truncated ALK kinase mRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least95% identical to a mutant ALK polypeptide of the invention or to thenucleic acid sequence of the deposited cDNAs that do, in fact, encode afusion polypeptide having ALK kinase activity. Such activity may besimilar, but not necessarily identical, to the activity of an EML4-ALKfusion protein disclosed herein (either the full-length protein, themature protein, or a protein fragment that retains kinase activity), asmeasured in a particular biological assay. For example, the kinaseactivity of ALK can be examined by determining its ability tophosphorylate one or more tyrosine containing peptide substrates, forexample, Insulin Receptor Substrate 1 or 2 (IRS1, IRS2), which aresubstrates for the ALK kinase.

Due to the degeneracy of the genetic code, one of ordinary skill in theart will immediately recognize that a large number of the nucleic acidmolecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequence of the deposited cDNAs or thenucleic acid sequences shown in FIGS. 2A-B (SEQ ID NOs: 2 and 19) willencode a mutant polypeptide having ALK activity. In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidethat retains ALK kinase activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247: 1306-1310 (1990), which describes two main approaches for studyingthe tolerance of an amino acid sequence to change. The first methodrelies on the process of evolution, in which mutations are eitheraccepted or rejected by natural selection. The second approach usesgenetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality. These studies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Skilledartisans familiar with such techniques also appreciate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie et al., supra., and the references cited therein.

Methods for DNA sequencing that are well known and generally availablein the art may be used to practice any polynucleotide embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

Polynucleotide sequences encoding a mutant ALK polypeptide of theinvention may be extended utilizing a partial nucleotide sequence andemploying various methods known in the art to detect upstream sequencessuch as promoters and regulatory elements. For example, one method thatmay be employed, “restriction-site” PCR, uses universal primers toretrieve unknown sequence adjacent to a known locus (Sarkar, G., PCRMethods Applic. 2:318-322 (1993)). In particular, genomic DNA is firstamplified in the presence of primer to linker sequence and a primerspecific to the known region. Exemplary primers are those described inExample 2 herein. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16:8186 (1988)). The primers may be designed using OLIGO 4.06Primer Analysis software (National Biosciences Inc., Plymouth, Minn.),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic.1:111-119 (1991)). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR. Another method which may be used to retrieveunknown sequences is that described in Parker et al., Nucleic Acids Res.19: 3055-3060 (1991)). Additionally, one may use PCR, nested primers,and PROMOTERFINDER® libraries to walk in genomic DNA (Clontech, PaloAlto, Calif.). This process avoids the need to screen libraries and isuseful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into the 5′ and 3′ non-transcribedregulatory regions.

Capillary electrophoresis systems, which are commercially available, maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER™ and SEQUENCE NAVIGATOR™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA that might be present in limited amounts in aparticular sample.

C. Vectors and Host Cells.

The present invention also provides recombinant vectors that comprise anisolated polynucleotide of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof recombinant EML4-ALK polypeptides or fragments thereof by recombinanttechniques.

Recombinant constructs may be introduced into host cells usingwell-known techniques such infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host. In certain preferredembodiments in this regard, the vectors provide for specific expression,which may be inducible and/or cell type-specific. Particularly preferredamong such vectors are those inducible by environmental factors that areeasy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert comprising an EML4-ALK polynucleotide or of the inventionshould be operatively linked to an appropriate promoter, such as thephage lambda PL promoter, the E. coli lac, trp and tac promoters, theSV40 early and late promoters and promoters of retroviral LTRs, to namea few. Other suitable promoters are known to the skilled artisan. Theexpression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (UAA,UGA or UAG) appropriately positioned at the end of the polypeptide to betranslated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (1989)CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y, and Grant et al., Methods Enzymol. 153:516-544 (1997).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY (1986).

Transcription of DNA encoding an EML4-ALK fusion polypeptide of thepresent invention by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at basepairs 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein (e.g. a GST-fusion), and may include not only secretion signals,but also additional heterologous functional regions. For instance, aregion of additional amino acids, particularly charged amino acids, maybe added to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins.

EML4-ALK fusion polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Accordingly, in one embodiment, the invention provides a method forproducing a recombinant EML4-ALK fusion polypeptide by culturing arecombinant host cell (as described above) under conditions suitable forthe expression of the fusion polypeptide and recovering the polypeptide.Culture conditions suitable for the growth of host cells and theexpression of recombinant polypeptides from such cells are well known tothose of skill in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel F M et al., eds., Volume 2, Chapter 16, WileyInterscience.

D. Isolated Polypeptides.

The invention also provides, in part, isolated mutant ALK kinasepolypeptides and fragments thereof. In one embodiment, the inventionprovides an isolated polypeptide comprising an amino acid sequence atleast 95% identical to a sequence selected from the group consisting of:

(a) an amino acid sequence encoding an EML4-ALK fusion polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18;

(b) an amino acid sequence encoding an EML4-ALK fusion polypeptidecomprising the N-terminal amino acid sequence of EML-4 (residues 1-222of SEQ ID NO: 3 or residues 1-495 of SEQ ID NO: 3) and the kinase domainof ALK (residues 1116-1383 of SEQ ID NO: 5); and

(c) an amino acid sequence encoding a polypeptide comprising at leastsix contiguous amino acids encompassing the fusion junction (residues233-234 of SEQ ID NO: 1 or residues 495-496 of SEQ ID NO: 18) of anEML4-ALK fusion polypeptide.

In one preferred embodiment, recombinant mutant ALK polypeptides of theinvention are provided, which may be produced using a recombinant vectoror recombinant host cell as described above.

It will be recognized in the art that some amino acid sequences of anEML4-ALK fusion polypeptide or truncated active ALK kinase polypeptidecan be varied without significant effect of the structure or function ofthe mutant protein. If such differences in sequence are contemplated, itshould be remembered that there will be critical areas on the proteinwhich determine activity (e.g. the kinase domain of ALK). In general, itis possible to replace residues that form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of an EML4-ALK fusionpolypeptide that retain substantial ALK kinase activity or that includeother regions of EML-4 or ALK proteins, such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions (for example, substituting onehydrophilic residue for another, but not strongly hydrophilic forstrongly hydrophobic as a rule). Small changes or such “neutral” aminoacid substitutions will generally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and lie;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGin, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. Examples of conservative amino acidsubstitutions known to those skilled in the art are: Aromatic:phenylalanine tryptophan tyrosine; Hydrophobic: leucine isoleucinevaline; Polar: glutamine asparagines; Basic: arginine lysine histidine;Acidic: aspartic acid glutamic acid; Small: alanine serine threoninemethionine glycine. As indicated in detail above, further guidanceconcerning which amino acid changes are likely to be phenotypicallysilent (i.e., are not likely to have a significant deleterious effect ona function) can be found in Bowie et al., Science 247, supra.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of an EML4-ALK fusion polypeptide of theinvention can be substantially purified by the one-step method describedin Smith and Johnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include the EML4-ALK fusionpolypeptides of FIGS. 2A-B (SEQ ID NOs: 1 and 18) (whether or notincluding a leader sequence), an amino acid sequence encoding anEML4-ALK fusion polypeptide comprising the N-terminal amino acidsequence of EML-4 (residues 1-222 of SEQ ID NO: 3 or residues 1-495 ofSEQ ID NO: 3) and the kinase domain of ALK (residues 1116-1383 of SEQ IDNO: 5), and an amino acid sequence encoding a polypeptide comprising atleast six contiguous amino acids encompassing the fusion junction(residues 233-234 of SEQ ID NO: 1 or residues 495-496 of SEQ ID NO: 18)of an EML4-ALK fusion polypeptide (see also FIGS. 1A-B, bottom panel),as well as polypeptides that have at least 90% similarity, preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an EML4-ALK fusionpolypeptide of the invention is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid sequence of the mutantALK polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

When using Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

An EML4-ALK fusion polypeptide of the present invention may be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns, for example, using methods well known to those ofskill in the art.

As further described in detail below, the polypeptides of the presentinvention can also be used to generate fusion polypeptide specificreagents, such as polyclonal and monoclonal antibodies, or truncatedpolypeptide specific reagents, which are useful in assays for detectingmutant ALK polypeptide expression as described below, or as agonists andantagonists capable of enhancing or inhibiting the function/activity ofthe mutant ALK protein. Further, such polypeptides can be used in theyeast two-hybrid system to “capture” EML4-ALK fusion polypeptide ortruncated ALK kinase polypeptide binding proteins, which are alsocandidate agonist and antagonist according to the present invention. Theyeast two hybrid system is described in Fields and Song, Nature340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention,for example, an epitope comprising the fusion junction of an EML4-ALKfusion polypeptide (see FIGS. 1A-B, bottom panel). The epitope of thispolypeptide portion is an immunogenic or antigenic epitope of apolypeptide of the invention. An “immunogenic epitope” is defined as apart of a protein that elicits an antibody response when the wholeprotein is the immunogen. These immunogenic epitopes are believed to beconfined to a few loci on the molecule. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).The production of fusion polypeptide-specific antibodies of theinvention is described in further detail below.

The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect a mimicked protein, and antibodies todifferent peptides may be used for tracking the fate of various regionsof a protein precursor that undergoes post-translational processing. Thepeptides and anti-peptide antibodies may be used in a variety ofqualitative or quantitative assays for the mimicked protein, forinstance in competition assays since it has been shown that even shortpeptides (e.g., about 9 amino acids) can bind and displace the largerpeptides in immunoprecipitation assays. See, for instance, Wilson etal., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies of theinvention also are useful for purification of the mimicked protein, forinstance, by adsorption chromatography using methods well known in theart. Immunological assay formats are described in further detail below.

Recombinant mutant ALK polypeptides are also within the scope of thepresent invention, and may be producing using polynucleotides of theinvention, as described in Section B above. For example, the inventionprovides, in part, a method for producing a recombinant EML4-ALK fusionpolypeptide by culturing a recombinant host cell (as described above)under conditions suitable for the expression of the fusion polypeptideand recovering the polypeptide. Culture conditions suitable for thegrowth of host cells and the expression of recombinant polypeptides fromsuch cells are well known to those of skill in the art.

E. Mutant-Specific Reagents

Mutant ALK polypeptide-specific reagents useful in the practice of thedisclosed methods include, among others, fusion polypeptide specificantibodies and AQUA peptides (heavy-isotope labeled peptides)corresponding to, and suitable for detection and quantification of,EML4-ALK fusion polypeptide expression in a biological sample from acancer, such as a mammalian solid sarcoma or carcinoma tumor. Alsouseful are truncation-specific reagents, such as antibodies, AQUApeptides, or nucleic acid probes, suitable for detecting the presence orabsence of a truncated ALK kinase polynucleotide or polypeptide of theinvention. A fusion polypeptide-specific reagent is any reagent,biological or chemical, capable of specifically binding to, detectingand/or quantifying the presence/level of expressed EML4-ALK fusionpolypeptide in a biological sample. The term includes, but is notlimited to, the preferred antibody and AQUA peptide reagents discussedbelow, and equivalent reagents are within the scope of the presentinvention.

Antibodies.

Reagents suitable for use in practice of the methods of the inventioninclude an EML4-ALK fusion polypeptide-specific antibody and a TFG-ALKfusion polypeptide-specific antibody. A fusion-specific antibody of theinvention is an isolated antibody or antibodies that specificallybind(s) an EML4-ALK fusion polypeptide of the invention (e.g. SEQ IDNO: 1) but does not substantially bind either wild type EML-4 or wildtype ALK, or specifically bind(s) a TFG-ALK fusion polypeptide describedherein (e.g. SEQ ID NO: 20) but does not substantially bind either wildtype TFG or wild type ALK. Other suitable reagents includeepitope-specific antibodies that specifically bind to an epitope in theextracelluar domain of wild type ALK protein sequence (which domain isnot present in the truncated, active ALK kinase disclosed herein), andare therefore capable of detecting the presence (or absence) of wildtype ALK in a sample.

Human EML4-ALK or TFG-ALK fusion polypeptide-specific antibodies mayalso bind to highly homologous and equivalent epitopic peptide sequencesin other mammalian species, for example murine or rabbit, and viceversa. Antibodies useful in practicing the methods of the inventioninclude (a) monoclonal antibodies, (b) purified polyclonal antibodiesthat specifically bind to the target polypeptide (e.g. the fusionjunction of EML4-ALK fusion polypeptide (see FIGS. 1A-B, bottom panel)or TFG-ALK fusion polypeptide (see FIG. 1C, bottom panel), (c)antibodies as described in (a)-(b) above that bind equivalent and highlyhomologous epitopes or phosphorylation sites in other non-human species(e.g. mouse, rat), and (d) fragments of (a)-(c) above that bind to theantigen (or more preferably the epitope) bound by the exemplaryantibodies disclosed herein.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Theantibodies may be monoclonal or polyclonal and may be of any species oforigin, including (for example) mouse, rat, rabbit, horse, or human, ormay be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol.26:403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851(1984); Neuberger et al., Nature 312:604 (1984)). The antibodies may berecombinant monoclonal antibodies produced according to the methodsdisclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No.4,816,567 (Cabilly et al.) The antibodies may also be chemicallyconstructed specific antibodies made according to the method disclosedin U.S. Pat. No. 4,676,980 (Segel et al.)

The preferred epitopic site of an EML4-ALK fusion polypeptide specificantibody of the invention is a peptide fragment consisting essentiallyof about 11 to 17 amino acids of a human EML4-ALK fusion polypeptidesequence (SEQ ID NOs: 1 and 18) which fragment encompasses the fusionjunction (which occurs at residues 233-234 in the short variant fusionprotein and residues 495-496 in the long variant fusion protein (seeFIGS. 1A-B (bottom panel)). It will be appreciated that antibodies thatspecifically binding shorter or longer peptides/epitopes encompassingthe fusion junction of an EML4-ALK fusion polypeptide are within thescope of the present invention.

Similarly, the preferred epitopic site of a TFG-ALK fusion polypeptidespecific antibody useful in the practice of the disclosed methods is apeptide fragment consisting essentially of about 11 to 17 amino acids ofthe human TFG-ALK fusion polypeptide sequence (SEQ ID NO: 20), whichfragment encompasses the fusion junction (which occurs at residues137-138 (see FIG. 1C (bottom panel)).

The invention is not limited to use of antibodies, but includesequivalent molecules, such as protein binding domains or nucleic acidaptamers, which bind, in a fusion-protein or truncated-protein specificmanner, to essentially the same epitope to which an EML4-ALK or TFG-ALKfusion polypeptide-specific antibody useful in the methods of theinvention binds. See, e.g., Neuberger et al., Nature 312: 604 (1984).Such equivalent non-antibody reagents may be suitably employed in themethods of the invention further described below.

Polyclonal antibodies useful in practicing the methods of the inventionmay be produced according to standard techniques by immunizing asuitable animal (e.g., rabbit, goat, etc.) with an antigen encompassinga desired fusion-protein specific epitope (e.g. the fusion junction ofan ALK fusion protein described herein), collecting immune serum fromthe animal, and separating the polyclonal antibodies from the immuneserum, and purifying polyclonal antibodies having the desiredspecificity, in accordance with known procedures. The antigen may be asynthetic peptide antigen comprising the desired epitopic sequence,selected and constructed in accordance with well-known techniques. See,e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow &Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods InEnzymology, 201:264-283 (1991); Merrifield, J. Am. Chem. Soc. 85:21-49(1962)). Polyclonal antibodies produced as described herein may bescreened and isolated as further described below.

Monoclonal antibodies may also be beneficially employed in the methodsof the invention, and may be produced in hybridoma cell lines accordingto the well-known technique of Kohler and Milstein. Nature 265: 495-97(1975); Kohler and Milstein, Eur. J. Immunol. 6:511 (1976); see also,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989).Monoclonal antibodies so produced are highly specific, and improve theselectivity and specificity of assay methods provided by the invention.For example, a solution containing the appropriate antigen (e.g. asynthetic peptide comprising the fusion junction of EML4-ALK fusionpolypeptide) may be injected into a mouse and, after a sufficient time(in keeping with conventional techniques), the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells, typically in the presence of polyethyleneglycol, to produce hybridoma cells. Rabbit fusion hybridomas, forexample, may be produced as described in U.S. Pat. No. 5,675,063, K.Knight, Issued Oct. 7, 1997. The hybridoma cells are then grown in asuitable selection media, such as hypoxanthine-aminopterin-thymidine(HAT), and the supernatant screened for monoclonal antibodies having thedesired specificity, as described below. The secreted antibody may berecovered from tissue culture supernatant by conventional methods suchas precipitation, ion exchange or affinity chromatography, or the like.

Monoclonal Fab fragments may also be produced in Escherichia coli byrecombinant techniques known to those skilled in the art. See, e.g., W.Huse, Science 246:1275-81 (1989); Mullinax et al., Proc. Nat'l Acad.Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype arepreferred for a particular application, particular isotypes can beprepared directly, by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82:8653 (1985); Spira et al., J. Immunol. Methods, 74:307 (1984)). Theantigen combining site of the monoclonal antibody can be cloned by PCRand single-chain antibodies produced as phage-displayed recombinantantibodies or soluble antibodies in E. coli (see, e.g., ANTIBODYENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)

Further still, U.S. Pat. No. 5,194,392, Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) that is a topological equivalent of theepitope (i.e., a “mimotope”) that is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, this method involves detecting or determining a sequence ofmonomers that is a topographical equivalent of a ligand that iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971, Houghten et al. (1996)discloses linear C₁-C-alkyl peralkylated oligopeptides and sets andlibraries of such peptides, as well as methods for using sucholigopeptide sets and libraries for determining the sequence of aperalkylated oligopeptide that preferentially binds to an acceptormolecule of interest. Thus, non-peptide analogs of the epitope-bearingpeptides of the invention also can be made routinely by these methods.

Antibodies useful in the methods of the invention, whether polyclonal ormonoclonal, may be screened for epitope and fusion protein specificityaccording to standard techniques. See, e.g. Czernik et al., Methods inEnzymology, 201:264-283 (1991). For example, the antibodies may bescreened against a peptide library by ELISA to ensure specificity forboth the desired antigen and, if desired, for reactivity only with, e.g.an EML4-ALK fusion polypeptide of the invention and not with wild typeEML-4 or wild type ALK. The antibodies may also be tested by Westernblotting against cell preparations containing target protein to confirmreactivity with the only the desired target and to ensure no appreciablebinding to other fusion proteins involving ALK. The production,screening, and use of fusion protein-specific antibodies is known tothose of skill in the art, and has been described. See, e.g., U.S.Patent Publication No. 20050214301, Wetzel et al., Sep. 29, 2005.

Fusion polypeptide-specific antibodies useful in the methods of theinvention may exhibit some limited cross-reactivity with similar fusionepitopes in other fusion proteins or with the epitopes in wild typeEML-4, wild type TFG, and wild type ALK that form the fusion junction.This is not unexpected as most antibodies exhibit some degree ofcross-reactivity, and anti-peptide antibodies will often cross-reactwith epitopes having high homology or identity to the immunizingpeptide. See, e.g., Czernik, supra. Cross-reactivity with other fusionproteins is readily characterized by Western blotting alongside markersof known molecular weight. Amino acid sequences of cross-reactingproteins may be examined to identify sites highly homologous oridentical to the EML4-ALK or TFG-ALK fusion polypeptide sequence towhich the antibody binds. Undesirable cross-reactivity can be removed bynegative selection using antibody purification on peptide columns (e.g.selecting out antibodies that bind either wild type EML-4 and/or wildtype ALK).

EML4-ALK fusion polypeptide-specific antibodies of the invention (andTFG-ALK fusion polypeptide-specific antibodies) that are useful inpracticing the methods disclosed herein are ideally specific for humanfusion polypeptide, but are not limited only to binding the humanspecies, per se. The invention includes the production and use ofantibodies that also bind conserved and highly homologous or identicalepitopes in other mammalian species (e.g. mouse, rat, monkey). Highlyhomologous or identical sequences in other species can readily beidentified by standard sequence comparisons, such as using BLAST, with ahuman EML4-ALK fusion polypeptide sequence disclosed herein (SEQ ID NOs:1 and 18) or a human TFG-ALK fusion polypeptide sequence disclosedherein (SEQ ID NO: 20).

Antibodies employed in the methods of the invention may be furthercharacterized by, and validated for, use in a particular assay format,for example flow cytometry (FC), immunohistochemistry (IHC), and/orImmunocytochemistry (ICC). The use of ALK fusion polypeptide-specificantibodies in such methods is further described in Section F below.Antibodies may also be advantageously conjugated to fluorescent dyes(e.g. Alexa488, PE), or labels such as quantum dots, for use inmulti-parametric analyses along with other signal transduction(phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin)antibodies, as further described in Section F below.

In practicing the methods of the invention, the expression and/oractivity of wild type EML-4, wild type TFG, and/or wild type ALK in agiven biological sample may also be advantageously examined usingantibodies (either phospho-specific or total) for these wild typeproteins. For example, ALK total and phosphorylation-site specificantibodies are commercially available (see CELL SIGNALING TECHNOLOGY,INC., Beverly Mass., 2005/06 Catalogue, #'s 3341, 3342). Such antibodiesmay also be produced according to standard methods, as described above.The amino acid sequences of human EML-4, TFG, and ALK are published (seeFIGS. 3A and 4A-4C, and referenced SwissProt Accession Nos.), as are thesequences of these proteins from other species.

Detection of wild type EML-4, TFG, and wild type ALK expression and/oractivation, along with EML4-ALK and/or TFG-ALK fusion polypeptideexpression, in a biological sample (e.g. a tumor sample) can provideinformation on whether the fusion protein alone is driving the tumor, orwhether wild type ALK is also activated and driving the tumor. Suchinformation is clinically useful in assessing whether targeting thefusion protein or the wild type protein(s), or both, or is likely to bemost beneficial in inhibiting progression of the tumor, and in selectingan appropriate therapeutic or combination thereof. Antibodies specificfor the wild type ALK kinase extracellular domain, which is not presentin the truncated active ALK kinase disclosed herein, may be particularlyuseful for determining the presence/absence of the mutant ALK kinase.

It will be understood that more than one antibody may be used in thepractice of the above-described methods. For example, one or moreEML4-ALK fusion polypeptide-specific antibodies together with one ormore antibodies specific for another kinase, receptor, or kinasesubstrate that is suspected of being, or potentially is, activated in acancer in which EML4-ALK fusion polypeptide is expressed may besimultaneously employed to detect the activity of such other signalingmolecules in a biological sample comprising cells from such cancer.

Those of skill in the art will appreciate that EML4-ALK fusionpolypeptides of the present invention and the fusion junctionepitope-bearing fragments thereof described above can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These fusion proteins facilitate purification andshow an increased half-life in vivo. This has been shown, e.g., forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins (EPA 394,827; Traunecker etal., Nature 331: 84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericEML4-ALK fusion polypeptide alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

Heavy-Isotope Labeled Peptides (AQUA Peptides).

EML4-ALK or TFG-ALK fusion polypeptide-specific reagents useful in thepractice of the disclosed methods may also comprise heavy-isotopelabeled peptides suitable for the absolute quantification of expressedALK fusion polypeptide or truncated ALK kinase polypeptide in abiological sample. The production and use of AQUA peptides for theabsolute quantification of proteins (AQUA) in complex mixtures has beendescribed. See WO/03016861, “Absolute Quantification of Proteins andModified Forms Thereof by Multistage Mass Spectrometry,” Gygi et al. andalso Gerber et al. Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003) (theteachings of which are hereby incorporated herein by reference, in theirentirety).

The AQUA methodology employs the introduction of a known quantity of atleast one heavy-isotope labeled peptide standard (which has a uniquesignature detectable by LC-SRM chromatography) into a digestedbiological sample in order to determine, by comparison to the peptidestandard, the absolute quantity of a peptide with the same sequence andprotein modification in the biological sample. Briefly, the AQUAmethodology has two stages: peptide internal standard selection andvalidation and method development; and implementation using validatedpeptide internal standards to detect and quantify a target protein insample. The method is a powerful technique for detecting and quantifyinga given peptide/protein within a complex biological mixture, such as acell lysate, and may be employed, e.g., to quantify change in proteinphosphorylation as a result of drug treatment, or to quantifydifferences in the level of a protein in different biological states.

Generally, to develop a suitable internal standard, a particular peptide(or modified peptide) within a target protein sequence is chosen basedon its amino acid sequence and the particular protease to be used todigest. The peptide is then generated by solid-phase peptide synthesissuch that one residue is replaced with that same residue containingstable isotopes (¹³C, ¹⁵N). The result is a peptide that is chemicallyidentical to its native counterpart formed by proteolysis, but is easilydistinguishable by MS via a 7-Da mass shift. The newly synthesized AQUAinternal standard peptide is then evaluated by LC-MS/MS. This processprovides qualitative information about peptide retention byreverse-phase chromatography, ionization efficiency, and fragmentationvia collision-induced dissociation. Informative and abundant fragmentions for sets of native and internal standard peptides are chosen andthen specifically monitored in rapid succession as a function ofchromatographic retention to form a selected reaction monitoring(LC-SRM) method based on the unique profile of the peptide standard.

The second stage of the AQUA strategy is its implementation to measurethe amount of a protein or modified protein from complex mixtures. Wholecell lysates are typically fractionated by SDS-PAGE gel electrophoresis,and regions of the gel consistent with protein migration are excised.This process is followed by in-gel proteolysis in the presence of theAQUA peptides and LC-SRM analysis. (See Gerber et al. supra.) AQUApeptides are spiked in to the complex peptide mixture obtained bydigestion of the whole cell lysate with a proteolytic enzyme andsubjected to immunoaffinity purification as described above. Theretention time and fragmentation pattern of the native peptide formed bydigestion (e.g. trypsinization) is identical to that of the AQUAinternal standard peptide determined previously; thus, LC-MS/MS analysisusing an SRM experiment results in the highly specific and sensitivemeasurement of both internal standard and analyte directly fromextremely complex peptide mixtures.

Since an absolute amount of the AQUA peptide is added (e.g. 250 fmol),the ratio of the areas under the curve can be used to determine theprecise expression levels of a protein or phosphorylated form of aprotein in the original cell lysate. In addition, the internal standardis present during in-gel digestion as native peptides are formed, suchthat peptide extraction efficiency from gel pieces, absolute lossesduring sample handling (including vacuum centrifugation), andvariability during introduction into the LC-MS system do not affect thedetermined ratio of native and AQUA peptide abundances.

An AQUA peptide standard is developed for a known sequence previouslyidentified by the IAP-LC-MS/MS method within in a target protein. If thesite is modified, one AQUA peptide incorporating the modified form ofthe particular residue within the site may be developed, and a secondAQUA peptide incorporating the unmodified form of the residue developed.In this way, the two standards may be used to detect and quantify boththe modified an unmodified forms of the site in a biological sample.

Peptide internal standards may also be generated by examining theprimary amino acid sequence of a protein and determining the boundariesof peptides produced by protease cleavage. Alternatively, a protein mayactually be digested with a protease and a particular peptide fragmentproduced can then sequenced. Suitable proteases include, but are notlimited to, serine proteases (e.g. trypsin, hepsin), metallo proteases(e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin,carboxypeptidases, etc.

A peptide sequence within a target protein is selected according to oneor more criteria to optimize the use of the peptide as an internalstandard. Preferably, the size of the peptide is selected to minimizethe chances that the peptide sequence will be repeated elsewhere inother non-target proteins. Thus, a peptide is preferably at least about6 amino acids. The size of the peptide is also optimized to maximizeionization frequency. Thus, peptides longer than about 20 amino acidsare not preferred. The preferred ranged is about 7 to 15 amino acids. Apeptide sequence is also selected that is not likely to be chemicallyreactive during mass spectrometry, thus sequences comprising cysteine,tryptophan, or methionine are avoided.

A peptide sequence that does not include a modified region of the targetregion may be selected so that the peptide internal standard can be usedto determine the quantity of all forms of the protein. Alternatively, apeptide internal standard encompassing a modified amino acid may bedesirable to detect and quantify only the modified form of the targetprotein. Peptide standards for both modified and unmodified regions canbe used together, to determine the extent of a modification in aparticular sample (i.e. to determine what fraction of the total amountof protein is represented by the modified form). For example, peptidestandards for both the phosphorylated and unphosphorylated form of aprotein known to be phosphorylated at a particular site can be used toquantify the amount of phosphorylated form in a sample.

The peptide is labeled using one or more labeled amino acids (i.e. thelabel is an actual part of the peptide) or less preferably, labels maybe attached after synthesis according to standard methods. Preferably,the label is a mass-altering label selected based on the followingconsiderations: The mass should be unique to shift fragments massesproduced by MS analysis to regions of the spectrum with low background;the ion mass signature component is the portion of the labeling moietythat preferably exhibits a unique ion mass signature in MS analysis; thesum of the masses of the constituent atoms of the label is preferablyuniquely different than the fragments of all the possible amino acids.As a result, the labeled amino acids and peptides are readilydistinguished from unlabeled ones by the ion/mass pattern in theresulting mass spectrum. Preferably, the ion mass signature componentimparts a mass to a protein fragment that does not match the residuemass for any of the 20 natural amino acids.

The label should be robust under the fragmentation conditions of MS andnot undergo unfavorable fragmentation. Labeling chemistry should beefficient under a range of conditions, particularly denaturingconditions, and the labeled tag preferably remains soluble in the MSbuffer system of choice. The label preferably does not suppress theionization efficiency of the protein and is not chemically reactive. Thelabel may contain a mixture of two or more isotopically distinct speciesto generate a unique mass spectrometric patter at each labeled fragmentposition. Stable isotopes, such as ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, or ³⁴S, areamong preferred labels. Pairs of peptide internal standards thatincorporate a different isotope label may also be prepared. Preferredamino acid residues into which a heavy isotope label may be incorporatedinclude leucine, proline, valine, and phenylalanine.

Peptide internal standards are characterized according to theirmass-to-charge (m/z) ratio, and preferably, also according to theirretention time on a chromatographic column (e.g. an HPLC column).Internal standards that co-elute with unlabeled peptides of identicalsequence are selected as optimal internal standards. The internalstandard is then analyzed by fragmenting the peptide by any suitablemeans, for example by collision-induced dissociation (CID) using, e.g.,argon or helium as a collision gas. The fragments are then analyzed, forexample by multi-stage mass spectrometry (MS^(n)) to obtain a fragmention spectrum, to obtain a peptide fragmentation signature. Preferably,peptide fragments have significant differences in m/z ratios to enablepeaks corresponding to each fragment to be well separated, and asignature is that is unique for the target peptide is obtained. If asuitable fragment signature is not obtained at the first stage,additional stages of MS are performed until a unique signature isobtained.

Fragment ions in the MS/MS and MS³ spectra are typically highly specificfor the peptide of interest, and, in conjunction with LC methods, allowa highly selective means of detecting and quantifying a targetpeptide/protein in a complex protein mixture, such as a cell lysate,containing many thousands or tens of thousands of proteins. Anybiological sample potentially containing a target protein/peptide ofinterest may be assayed. Crude or partially purified cell extracts arepreferably employed. Generally, the sample has at least 0.01 mg ofprotein, typically a concentration of 0.1-10 mg/mL, and may be adjustedto a desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about10 femtomoles, corresponding to a target protein to bedetected/quantified is then added to a biological sample, such as a celllysate. The spiked sample is then digested with one or more protease(s)for a suitable time period to allow digestion. A separation is thenperformed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis,ion exchange chromatography, etc.) to isolate the labeled internalstandard and its corresponding target peptide from other peptides in thesample. Microcapillary LC is a preferred method.

Each isolated peptide is then examined by monitoring of a selectedreaction in the MS. This involves using the prior knowledge gained bythe characterization of the peptide internal standard and then requiringthe MS to continuously monitor a specific ion in the MS/MS or MS^(n)spectrum for both the peptide of interest and the internal standard.After elution, the area under the curve (AUC) for both peptide standardand target peptide peaks are calculated. The ratio of the two areasprovides the absolute quantification that can be normalized for thenumber of cells used in the analysis and the protein's molecular weight,to provide the precise number of copies of the protein per cell. Furtherdetails of the AQUA methodology are described in Gygi et al., and Gerberet al. supra

AQUA internal peptide standards (heavy-isotope labeled peptides) maydesirably be produced, as described above, to detect any quantify anyunique site (e.g. the fusion junction within a disclosed EML4-ALK fusionpolypeptide) within a mutant ALK polypeptide of the invention. Forexample, an AQUA phosphopeptide may be prepared that corresponds to thefusion junction sequence of an EML4-ALK fusion polypeptide (see FIGS.1A-B (bottom panel)) or that corresponds to the truncation point ofeither EML4, TFG, or ALK. Peptide standards for may be produced for theEML4-ALK or TFG-ALK fusion junction and such standards employed in theAQUA methodology to detect and quantify the fusion junction (i.e. thepresence of EML4-ALK fusion polypeptide) in a biological sample.

For example, an exemplary AQUA peptide of the invention comprises theamino acid sequence INQVYR (see FIG. 1, bottom panel), which correspondsto the three amino acids immediately flanking each side of the fusionjunction in EML4-ALK fusion polypeptide (see SEQ ID NO: 7). It will beappreciated that larger AQUA peptides comprising the fusion junctionsequence (and additional residues downstream or upstream of it) may alsobe constructed. Similarly, a smaller AQUA peptide comprising less thanall of the residues of such sequence (but still comprising the point offusion junction itself) may alternatively be constructed. Such larger orshorter AQUA peptides are within the scope of the present invention, andthe selection and production of preferred AQUA peptides may be carriedout as described above (see Gygi et al., Gerber et al., supra.).

Nucleic Acid Probes.

Fusion-specific reagents provided by the invention also include nucleicacid probes and primers suitable for detection of an EML4-ALKpolynucleotide or truncated ALK kinase polynucleotide, as described indetail in Section B above. Such probes desirable include, among others,breakpoint probes corresponding to both sides of the breakpoints inwild-type EML4 and/or wild-type ALK genes that produce the fusion. Thespecific use of such probes in assays such as fluorescence in-situhybridization (FISH) or polymerase chain reaction (PCR) amplification isdescribed in Section F below. Similar breakpoint probes may be preparedto detect the presence of TFG-ALK fusion polynucleotide (see FIG. 1C(SEQ ID NO: 21).

F. Diagnostic Applications & Assay Formats.

The methods of the invention may be carried out in a variety ofdifferent assay formats known to those of skill in the art.

Immunoassays.

Immunoassays useful in the practice of the methods of the invention maybe homogenous immunoassays or heterogeneous immunoassays. In ahomogeneous assay the immunological reaction usually involves a mutantALK kinase polypeptide-specific reagent (e.g. an EML4-ALK fusionpolypeptide-specific antibody), a labeled analyte, and the biologicalsample of interest. The signal arising from the label is modified,directly or indirectly, upon the binding of the antibody to the labeledanalyte. Both the immunological reaction and detection of the extentthereof are carried out in a homogeneous solution. Immunochemical labelsthat may be employed include free radicals, radio-isotopes, fluorescentdyes, enzymes, bacteriophages, coenzymes, and so forth. Semi-conductornanocrystal labels, or “quantum dots”, may also be advantageouslyemployed, and their preparation and use has been well described. Seegenerally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) andpatents cited therein.

In a heterogeneous assay approach, the reagents are usually thebiological sample, a mutant ALK kinase polypeptide-specific reagent(e.g., an EML4-ALK fusion-specific antibody), and suitable means forproducing a detectable signal. Biological samples as further describedbelow may be used. The antibody is generally immobilized on a support,such as a bead, plate or slide, and contacted with the sample suspectedof containing the antigen in a liquid phase. The support is thenseparated from the liquid phase and either the support phase or theliquid phase is examined for a detectable signal employing means forproducing such signal. The signal is related to the presence of theanalyte in the biological sample. Means for producing a detectablesignal include the use of radioactive labels, fluorescent labels, enzymelabels, quantum dots, and so forth. For example, if the antigen to bedetected contains a second binding site, an antibody which binds to thatsite can be conjugated to a detectable group and added to the liquidphase reaction solution before the separation step. The presence of thedetectable group on the solid support indicates the presence of theantigen in the test sample. Examples of suitable immunoassays are theradioimmunoassay, immunofluorescence methods, enzyme-linkedimmunoassays, and the like.

Immunoassay formats and variations thereof, which may be useful forcarrying out the methods disclosed herein, are well known in the art.See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc.,Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold etal., “Methods for Modulating Ligand-Receptor Interactions and theirApplication”); U.S. Pat. No. 4,659,678 (Forrest et al., “Immunoassay ofAntigens”); U.S. Pat. No. 4,376,110 (David et al., “Immunometric AssaysUsing Monoclonal Antibodies”). Conditions suitable for the formation ofreagent-antibody complexes are well known to those of skill in the art.See id. EML4-ALK fusion polypeptide-specific monoclonal antibodies maybe used in a “two-site” or “sandwich” assay, with a single hybridomacell line serving as a source for both the labeled monoclonal antibodyand the bound monoclonal antibody. Such assays are described in U.S.Pat. No. 4,376,110. The concentration of detectable reagent should besufficient such that the binding of EML4-ALK or TFG-ALK fusionpolypeptide is detectable compared to background.

Antibodies useful in the practice of the methods disclosed herein may beconjugated to a solid support suitable for a diagnostic assay (e.g.,beads, plates, slides or wells formed from materials such as latex orpolystyrene) in accordance with known techniques, such as precipitation.Antibodies or other ALK fusion polypeptide- or truncated ALK kinasepolypeptide-binding reagents may likewise be conjugated to detectablegroups such as radiolabels (e.g., ³⁵S, ²⁵I, ¹³¹I), enzyme labels (e.g.,horseradish peroxidase, alkaline phosphatase), and fluorescent labels(e.g., fluorescein) in accordance with known techniques.

Cell-based assays, such flow cytometry (FC), immuno-histochemistry(IHC), or immunofluorescence (IF) are particularly desirable inpracticing the methods of the invention, since such assay formats areclinically-suitable, allow the detection of mutant ALK kinasepolypeptide expression in vivo, and avoid the risk of artifact changesin activity resulting from manipulating cells obtained from, e.g. atumor sample in order to obtain extracts. Accordingly, in some preferredembodiment, the methods of the invention are implemented in aflow-cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence(IF) assay format.

Flow cytometry (FC) may be employed to determine the expression ofmutant ALK kinase polypeptide in a mammalian tumor before, during, andafter treatment with a drug targeted at inhibiting ALK kinase activity.For example, tumor cells from a bone marrow sample may be analyzed byflow cytometry for EML4-ALK or TFG-ALK fusion polypeptide expressionand/or activation, as well as for markers identifying cancer cell types,etc., if so desired. Flow cytometry may be carried out according tostandard methods. See, e.g. Chow et al., Cytometry (Communications inClinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, thefollowing protocol for cytometric analysis may be employed: fixation ofthe cells with 2% paraformaldehyde for 10 minutes at 37° C. followed bypermeabilization in 90% methanol for 30 minutes on ice. Cells may thenbe stained with the primary EML4-ALK or TFG-ALK fusionpolypeptide-specific antibody, washed and labeled with afluorescent-labeled secondary antibody. The cells would then be analyzedon a flow cytometer (e.g. a Beckman Coulter FC500) according to thespecific protocols of the instrument used. Such an analysis wouldidentify the level of expressed EML4-ALK or TFG-ALK fusion polypeptidein the tumor. Similar analysis after treatment of the tumor with anALK-inhibiting therapeutic would reveal the responsiveness of an ALKfusion polypeptide-expressing tumor to the targeted inhibitor of ALKkinase.

Immunohistochemical (IHC) staining may be also employed to determine theexpression and/or activation status of mutant ALK kinase polypeptide ina mammalian cancer (e.g. a solid tumor like NSCLC) before, during, andafter treatment with a drug targeted at inhibiting ALK kinase activity.IHC may be carried out according to well-known techniques. See, e.g.,ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., ColdSpring Harbor Laboratory (1988). Briefly, and by way of example,paraffin-embedded tissue (e.g. tumor tissue from a biopsy) is preparedfor immunohistochemical staining by deparaffinizing tissue sections withxylene followed by ethanol; hydrating in water then PBS; unmaskingantigen by heating slide in sodium citrate buffer; incubating sectionsin hydrogen peroxide; blocking in blocking solution; incubating slide inprimary anti-EML4-ALK or anti-TFG-ALK fusion polypeptide antibody andsecondary antibody; and finally detecting using ABC avidin/biotin methodaccording to manufacturer's instructions.

Immunofluorescence (IF) assays may be also employed to determine theexpression and/or activation status of mutant ALK kinase polypeptide ina mammalian cancer before, during, and after treatment with a drugtargeted at inhibiting ALK kinase activity. IF may be carried outaccording to well-known techniques. See, e.g., J. M. Polak and S. VanNoorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYALMICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37,BioScientific/Springer-Verlag. Briefly, and by way of example, patientsamples may be fixed in paraformaldehyde followed by methanol, blockedwith a blocking solution such as horse serum, incubated with the primaryantibody against EML4-ALK or TFG-ALK fusion polypeptide followed by asecondary antibody labeled with a fluorescent dye such as Alexa 488 andanalyzed with an epifluorescent microscope.

Antibodies employed in the above-described assays may be advantageouslyconjugated to fluorescent dyes (e.g. Alexa488, PE), or other labels,such as quantum dots, for use in multi-parametric analyses along withother signal transduction (e.g. EGFR, phospho-AKT, phospho-Erk 1/2)and/or cell marker (e.g. cytokeratin) antibodies.

A variety of other protocols, including enzyme-linked immunosorbentassay (ELISA), radio-immunoassay (RIA), and fluorescent-activated cellsorting (FACS), for measuring mutant ALK kinase polypeptide are known inthe art and provide a basis for diagnosing altered or abnormal levels ofEML4-ALK or TFG-ALK fusion polypeptide expression. Normal or standardvalues for these fusion polypeptide expression are established bycombining body fluids or cell extracts taken from normal mammaliansubjects, preferably human, with antibody to EML4-ALK or TFG-ALK fusionpolypeptide under conditions suitable for complex formation. The amountof standard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of EML4-ALK or TFG-ALKfusion polypeptide expressed in subject, control, and disease samplesfrom biopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

Peptide & Nucleic Acid Assays.

Similarly, AQUA peptides for the detection/quantification of expressedmutant ALK kinase polypeptide in a biological sample comprising cellsfrom a tumor may be prepared and used in standard AQUA assays, asdescribed in detail in Section E above. Accordingly, in some preferredembodiments of the methods of the invention, the ALK fusionpolypeptide-specific reagent comprises a heavy isotope labeledphosphopeptide (AQUA peptide) corresponding to a peptide sequencecomprising the fusion junction of an EML4-ALK fusion polypeptide orTFG-ALK fusion polypeptide, as described above in Section E.

Mutant ALK polypeptide-specific reagents useful in practicing themethods of the invention may also be mRNA, oligonucleotide or DNA probesthat can directly hybridize to, and detect, fusion or truncatedpolypeptide expression transcripts in a biological sample. Such probesare discussed in detail in Section B above. Briefly, and by way ofexample, formalin-fixed, paraffin-embedded patient samples may be probedwith a fluorescein-labeled RNA probe followed by washes with formamide,SSC and PBS and analysis with a fluorescent microscope. Also preferredare FISH probes, including breakpoint probes, that allow the fluorescentdetection of gene rearrangements, such as the EML4-ALK deletionmutations on chromosome 2 (see Example 6).

Polynucleotides encoding mutant ALK kinase polypeptide may also be usedfor diagnostic purposes. The polynucleotides which may be used includeoligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.The polynucleotides may be used to detect and quantitate gene expressionin biopsied solid tumor tissues in which expression of EML4-ALK orTFG-ALK fusion polypeptide or truncated active ALK kinase polypeptidemay be correlated with disease. For example, the diagnostic assay may beused to distinguish between absence, presence, and excess expression ofEML4-ALK or TFG-ALK fusion polypeptide, and to monitor regulation of ALKfusion polypeptide levels during therapeutic intervention.

In one preferred embodiment, hybridization with PCR probes which arecapable of detecting polynucleotide sequences, including genomicsequences, encoding an ALK fusion polypeptide or truncated ALK kinasepolypeptide, or closely related molecules, may be used to identifynucleic acid sequences that encode mutant ALK polypeptide. Theconstruction and use of such probes is described in Section B above. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., 10 unique nucleotides in the fusion junction, or a lessspecific region, e.g., the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding mutant ALK polypeptide, alleles, or relatedsequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe mutant ALK polypeptide encoding sequences. The hybridization probesof the subject invention may be DNA or RNA and derived from thenucleotide sequences of SEQ ID NOs: 2, 19, and 21, most preferablyencompassing the fusion junction (see FIGS. 1A-C, bottom panel and SEQID NOs: 7, 24, and 26), or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring EML-4, TFG,and ALK polypeptides, as further described in Section B above.

For example, an EML4-ALK fusion polynucleotide of the invention may beused in Southern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered ALK polypeptide expression. Such qualitative or quantitativemethods are well known in the art. In a particular aspect, thenucleotide sequences encoding a mutant ALK polypeptide of the inventionmay be useful in assays that detect activation or induction of variouscancers, including lung carcinomas. Mutant ALK polynucleotides may belabeled by standard methods, and added to a fluid or tissue sample froma patient under conditions suitable for the formation of hybridizationcomplexes. After a suitable incubation period, the sample is washed andthe signal is quantitated and compared with a standard value. If theamount of signal in the biopsied or extracted sample is significantlyaltered from that of a comparable control sample, the nucleotidesequences have hybridized with nucleotide sequences in the sample, andthe presence of altered levels of nucleotide sequences encoding EML4-ALKfusion polypeptide or truncated ALK kinase polypeptide in the sampleindicates the presence of the associated disease. Such assays may alsobe used to evaluate the efficacy of a particular therapeutic treatmentregimen in animal studies, in clinical trials, or in monitoring thetreatment of an individual patient.

In order to provide a basis for the diagnosis of disease characterizedby expression of mutant ALK kinase polypeptide, a normal or standardprofile for expression is established. This may be accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with a sequence, or a fragment thereof, whichencodes EML4-ALK or TFG-ALK fusion polypeptide, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

Additional diagnostic uses for mutant ALK polynucleotides of theinvention may involve the use of polymerase chain reaction (PCR), apreferred assay format that is standard to those of skill in the art.See, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition,Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). PCR oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′ to 3′) and another withantisense (3′ to 5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of an ALKfusion polypeptide or truncated ALK kinase polypeptide includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and standard curves onto which the experimental resultsare interpolated (Melby et al., J. Immunol. Methods, 159: 235-244(1993); Duplaa et al. Anal. Biochem. 229-236 (1993)). The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In another embodiment of the invention, the mutant ALK polynucleotidesof the invention, as well the adjacent genomic region proximal anddistal to them, may be used to generate hybridization probes that areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well-known techniques. Such techniquesinclude fluorescence in-situ hybridization (FISH), FACS, or artificialchromosome constructions, such as yeast artificial chromosomes,bacterial artificial chromosomes, bacterial P1 constructions or singlechromosome cDNA libraries, as reviewed in Price, C. M., Blood Rev.7:127-134 (1993), and Trask, B. J., Trends Genet. 7:149-154 (1991).

In one preferred embodiment, FISH is employed (as described in Verma etal. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork, N.Y. (1988)) and may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in the 1994 Genome Issue of Science (265: 1981f).Correlation between the location of the gene encoding EML4-ALK orTFG-ALK fusion polypeptide or truncated active ALK kinase polypeptide ona physical chromosomal map and a specific disease, or predisposition toa specific disease, may help delimit the region of DNA associated withthat genetic disease. The nucleotide sequences of the subject inventionmay be used to detect differences in gene sequences between normal,carrier, or affected individuals. Dual-color breakpoint FISH probes, forexample, can be employed to detect the presence or absence of mutantEML-4, TFG, and/or ALK genes in a sample.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11 q22-23 (Gatti et al.,Nature 336:577-580 (1988)), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

Other suitable methods for nucleic acid detection, such as minorgroove-binding conjugated oligonucleotide probes (see, e.g. U.S. Pat.No. 6,951,930, “Hybridization-Triggered Fluorescent Detection of NucleicAcids”) are known to those of skill in the art.

It shall be understood that all of the methods (e.g., PCR and FISH) thatdetect EML4-ALK polynucleotides of the invention may be combined withother methods that detect either mutant ALK polynucleotides or mutantALK polypeptides. For example, detection of a EML4-ALK polynucleotide inthe genetic material of a biological sample (e.g., in a circulatingtumor cell) may be followed by Western blotting analysis orimmuno-histochemistry (IHC) analysis of the proteins of the sample todetermine if the EML4-ALK polynucleotide was actually expressed as aEML4-ALK polypeptide in the biological sample. Such Western blotting orIHC analyses may be performed using an antibody that specifically bindsto the polypeptide encoded by the detected EML4-ALK polynucleotide, orthe analyses may be performed using antibodies that specifically bindeither to full length EML4 (e.g., bind to the N-terminus of the protein)or to full length ALK (e.g., bind an epitope in the kinase domain ofALK). Such assays are known in the art (see, e.g., U.S. Pat. No.7,468,252).

In another example, the CISH technology of Dako allows chromatogenicin-situ hybridization with immuno-histochemistry on the same tissuesection.

Biological Samples.

Biological samples useful in the practice of the methods of theinvention may be obtained from any mammal in which a cancercharacterized by the expression of an EML4-ALK or TFG-ALK fusionpolypeptide is present or developing. In one embodiment, the mammal is ahuman, and the human may be a candidate for an ALK-inhibitingtherapeutic for the treatment of a cancer, e.g. NSCLC. The humancandidate may be a patient currently being treated with, or consideredfor treatment with, a ALK kinase inhibitor, such as WHI-131 and/orWHI-154. In another embodiment, the mammal is large animal, such as ahorse or cow, while in other embodiments, the mammal is a small animal,such as a dog or cat, all of which are known to develop cancers,including lung carcinomas.

Any biological sample comprising cells (or extracts of cells) from amammalian cancer is suitable for use in the methods of the invention. Inthe case of EML-ALK fusion polypeptide, any cancer, whether solid ornon-solid, will be suitable. In the case of TFG-ALK, solid tumors arewithin the scope of the methods of the invention. For example, thebiological sample may comprise cells obtained from an effusion, such asa pleural effusion. Pleural effusions (liquid that forms outside thelung in the thoracic cavity and which contains cancerous cells) areknown to form in many patients with advanced lung cancer (includingNSCLC), and the presence of such effusion is predictive of a pooroutcome and short survival time. Standard techniques for obtainingpleural effusion samples have been described and are well known in theart (see Sahn, Clin Chest Med. 3(2): 443-52 (1982)). Circulating tumorcells may also be obtained from serum using tumor markers, cytokeratinprotein markers or other methods of negative selection as described (seeMa et al., Anticancer Res. 23(1A): 49-62 (2003)). Serum and bone marrowsamples may be particularly preferred for patients with leukemia. Forcancers involving solid tumors, such as sarcomas and carcinomas, thebiological sample may comprise cells obtained from a tumor biopsy, whichmaybe be obtained according to standard clinical techniques. Forexample, aberrant expression of ALK has been observed in a spectrum ofcancers including neuroblastomas and neuroectodermal cancer. See, e.g.,Pulford et al., supra. The TFG-ALK translocation mutant, however, hasonly been described in lymphoma and not previously observed in solidtumors.

A biological sample may comprise cells (or cell extracts) from a cancerin which an ALK fusion polypeptide is expressed and/or activated butwild type ALK kinase is not. Alternatively, the sample may comprisecells from a cancer in which both the mutant ALK polypeptide and wildtype ALK kinase are expressed and/or activated, or in which wild typeALK kinase and/or EML-4 and/or TFG are expressed and/or active, butmutant ALK polypeptide is not.

In some embodiments, the biological sample comprises circulating tumorcells. Circulating tumor cells (“CTCs”) may be purified, for example,using the kits and reagents sold under the trademarks Vita-Assays™,Vita-Cap™, and CellSearch® (commercially available from Vitatex, LLC (aJohnson and Johnson corporation). Other methods for isolating CTCs aredescribed (see, for example, PCT Publication No. WO/2002/020825,Cristofanilli et al., New Engl. J. of Med. 351 (8):781-791 (2004), andAdams et al., J. Amer. Chem. Soc. 130(27): 8633-8641 (July 2008)). In aparticular embodiment, a circulating tumor cell (“CTC”) may be isolatedand identified as having originated from the lung.

Accordingly, the invention provides a method for isolating a CTC, andthen screening the CTC one or more assay formats to identify thepresence of a mutant EML4-ALK polypeptide or polynucleotide of theinvention in the CTC. Some non-limiting assay formats include Westernblotting analysis, flow-cytometry (FC), immuno-histochemistry (IHC),immuno-fluorescence (IF), fluorescence in situ hybridization (FISH) andpolymerase chain reaction (PCR). A CTC from a patient that is identifiedas comprising a EML4-ALK polypeptide or polynucleotide of the inventionmay indicate that the patient's originating cancer (e.g., a lung cancersuch as a non-small cell lung cancer) is likely to respond to acomposition comprising at least ALK-inhibiting therapeutic.

Cellular extracts of the foregoing biological samples may be prepared,either crude or partially (or entirely) purified, in accordance withstandard techniques, and used in the methods of the invention.Alternatively, biological samples comprising whole cells may be utilizedin preferred assay formats such as immunohistochemistry (IHC), flowcytometry (FC), immunofluorescence (IF), and fluorescence in situhybridization (FISH) as further described above. Such whole-cell assaysare advantageous in that they minimize manipulation of the tumor cellsample and thus reduce the risks of altering the in vivosignaling/activation state of the cells and/or introducing artifactsignals. Whole cell assays are also advantageous because theycharacterize expression and signaling only in tumor cells, rather than amixture of tumor and normal cells.

In practicing the disclosed method for determining whether a compoundinhibits progression of a tumor characterized by an EML4-ALK or TFG-ALKfusion gene and/or fusion polypeptide, biological samples comprisingcells from mammalian bone marrow transplant models or xenografts mayalso be advantageously employed. Preferred xenografts (or transplantrecipients) are small mammals, such as mice, harboring human tumors thatexpress a mutant ALK kinase polypeptide. Xenografts harboring humantumors are well known in the art (see Kal, Cancer Treat Res. 72: 155-69(1995)) and the production of mammalian xenografts harboring humantumors is well described (see Winograd et al., In Vivo. 1(1): 1-13(1987)). Similarly the generation and use of bone marrow transplantmodels is well described (see, e.g., Schwaller, et al., EMBO J.17:5321-333 (1998); Kelly et al., Blood 99: 310-318 (2002)). By “cancercharacterized by” an EML4-ALK or TFG-ALK fusion polynucleotide and/orfusion polypeptide is meant a cancer in which such mutant ALK geneand/or expressed polypeptide are present, as compared to a cancer inwhich such fusion gene and/or fusion polypeptide are not present.

In assessing mutant ALK polynucleotide presence or polypeptideexpression in a biological sample comprising cells from a mammaliancancer tumor, a control sample representing a cell in which suchtranslocation and/or fusion protein do not occur may desirably beemployed for comparative purposes. Ideally, the control sample comprisescells from a subset of the particular cancer (e.g. NSCLC) that isrepresentative of the subset in which the mutation (e.g. EML4-ALKdeletion mutation) does not occur and/or the fusion polypeptide is notexpressed. Comparing the level in the control sample versus the testbiological sample thus identifies whether the mutant ALK polynucleotideand/or polypeptide is/are present. Alternatively, since EML4-ALK and/orTFG-ALK fusion polynucleotide and/or polypeptide may not be present inthe majority of cancers, any tissue that similarly does not express suchmutant ALK polypeptide (or harbor the mutant polynucleotide) may beemployed as a control.

The methods described below will have valuable diagnostic utility forcancers characterized by mutant ALK polynucleotide and/or polypeptide,and treatment decisions pertaining to the same. For example, biologicalsamples may be obtained from a subject that has not been previouslydiagnosed as having a cancer characterized by an EML4-ALK deletionmutation and/or fusion polypeptide, nor has yet undergone treatment forsuch cancer, and the method is employed to diagnostically identify atumor in such subject as belonging to a subset of tumors (e.g. NSCLC) inwhich EML4-ALK fusion polynucleotide and/or polypeptide is presentand/or expressed. The methods of the invention may also be employed tomonitor the progression or inhibition of a mutant ALK kinasepolypeptide-expressing cancer following treatment of a subject with acomposition comprising an ALK kinase-inhibiting therapeutic orcombination of therapeutics.

Such diagnostic assay may be carried out subsequent to or prior topreliminary evaluation or surgical surveillance procedures. Theidentification method of the invention may be advantageously employed asa diagnostic to identify patients having cancer, such as NSCLC, drivenby the EML4-ALK and/or TFG-ALK fusion protein(s) or by truncated ALKkinase, which patients would be most likely to respond to therapeuticstargeted at inhibiting ALK kinase activity, such as WHI-131 and/orWHI-154 or their analogues. The ability to select such patients wouldalso be useful in the clinical evaluation of efficacy of futureALK-targeted therapeutics as well as in the future prescription of suchdrugs to patients.

Diagnostics.

The ability to selectively identify cancers in which an EML4-ALK and/orTFG-ALK fusion polynucleotide and/or fusion polypeptide is/are presentenables important new methods for accurately identifying such tumors fordiagnostic purposes, as well as obtaining information useful indetermining whether such a tumor is likely to respond to aALK-inhibiting therapeutic composition, or likely to be partially orwholly non-responsive to an inhibitor targeting a different kinase whenadministered as a single agent for the treatment of the caner.

Accordingly, in one embodiment, the invention provides a method fordetecting the presence of a mutant ALK polynucleotide and/or its encodedmutant ALK polypeptide in a biological sample from a mammalian cancer,said method comprising the steps of:

(a) obtaining a biological sample from a mammalian cancer; and

(b) utilizing at least one reagent that detects a fusion polynucleotide,or its encoded fusion polypeptide, comprising part of ALK with part of asecondary protein to determine whether an ALK mutant polynucleotideand/or its encoded mutant ALK polypeptide is present in said biologicalsample.

In some preferred embodiments the cancer is a solid tumor sarcoma orcarcinoma, while in one embodiment the carcinoma is a lung carcinoma,such as NSCLC. In another preferred embodiment the mutant ALKpolypeptide is a fusion polypeptide comprising residues 1116-1383 of ALK(SEQ ID NO: 5) with a portion of said secondary protein. In anotherpreferred embodiment, the secondary protein is selected from the groupconsisting of EML-4 (SEQ ID NO: 3) and TRK-Fused Gene (TFG) protein (SEQID NO: 22). In still another preferred embodiment, the fusionpolypeptide comprises residues 1-222 or residues 1-495 of EML-4 (SEQ IDNO: 3) or residues 1-138 of TFG (SEQ ID NO: 22).

In other preferred embodiments, the fusion polynucleotide comprises anEML4-ALK fusion polynucleotide (SEQ ID NOs: 2 or 19) or a TFG-ALK fusionpolynucleotide (SEQ ID NO: 21), while in still another embodiment thefusion polypeptide comprises an EML4-ALK fusion polypeptide (SEQ ID NOs:1 or 18) or a TFG-ALK fusion polypeptide (SEQ ID NO: 20). In yet anotherpreferred embodiment, the fusion polynucleotide is an EML4-ALK fusionpolynucleotide or polypeptide described above.

In more preferred embodiments, the method employs a reagent thatcomprises an EML4-ALK fusion polynucleotide and/or at least one EML4-ALKfusion polypeptide-specific reagent (antibody or AQUA peptide), asdescribed above. In some preferred embodiments, the reagent comprises anisolated reagent that specifically binds to or detects a TFG-ALK fusionpolypeptide (SEQ ID NO: 20) or TFG-ALK fusion polynucleotide (SEQ ID NO:21), but does not bind to or detect either wild type TFG or wild typeALK. In other preferred embodiments, the reagent is a polymerase chainreaction (PCR) probe or a fluorescence in situ hybridization (FISH)probe. Certain preferred embodiments employ a heavy isotope labeled(AQUA) peptide that comprises the amino acid sequence of the fusionjunction of TFG-ALK fusion polypeptide or truncation point withinwild-type ALK.

In some preferred embodiments, the diagnostic methods of the inventionare implemented in a flow-cytometry (FC), immuno-histochemistry (IHC),or immuno-fluorescence (IF) assay format, as described above. In anotherpreferred embodiment, the activity of the EML4-ALK or TFG-ALK fusionpolypeptide is detected. In other preferred embodiments, the diagnosticmethods of the invention are implemented in a fluorescence in situhybridization (FISH) or polymerase chain reaction (PCR) assay format, asdescribed above.

The invention further provides a method for determining whether acompound inhibits the progression of a cancer characterized by anEML4-ALK or TFG-ALK fusion polynucleotide and/or fusion polypeptide,said method comprising the step of determining whether said compoundinhibits the expression and/or activity of said EML4-ALK or TFG-ALKfusion polypeptide in said cancer. In one preferred embodiment,inhibition of expression and/or activity of the ALK fusion polypeptideis determined using at least one reagent that detects an EML4-ALK fusionpolynucleotide or polypeptide of the invention and/or a TFG-ALK fusionpolynucleotide or polypeptide described herein. Compounds suitable forinhibition of ALK kinase activity are discussed in more detail inSection G below.

Mutant ALK polynucleotide probes and polypeptide-specific reagentsuseful in the practice of the methods of the invention are described infurther detail in sections B and D above. In one preferred embodiment,the ALK fusion polypeptide-specific reagent comprises a fusionpolypeptide-specific antibody. In another preferred embodiment, thefusion polypeptide-specific reagent comprises a heavy-isotope labeledphosphopeptide (AQUA peptide) corresponding to the fusion junction of anALK fusion polypeptide (see FIGS. 1A-C (bottom panel)). In yet anotherpreferred embodiment, the fusion polynucleotide-specific reagentcomprises a FISH probe corresponding to the fusion junction of an ALKfusion gene and/or breakpoints of wild type EML4, TFG, or ALK genes.

The methods of the invention described above may also optionallycomprise the step of determining the level of expression or activationof other kinases, such as wild type ALK and EGFR, or other downstreamsignaling molecules in said biological sample. Profiling both ALK fusionpolypeptide expression/activation and expression/activation of otherkinases and pathways in a given biological sample can provide valuableinformation on which kinase(s) and pathway(s) is/are driving thedisease, and which therapeutic regime is therefore likely to be of mostbenefit.

Compound Screening.

The discovery of the novel EML4-ALK fusion polypeptides described hereinalso enables the development of new compounds that inhibit the activityof this mutant ALK protein, particularly its ALK kinase activity.Accordingly, the invention also provides, in part, a method fordetermining whether a compound inhibits the progression of a cancercharacterized by an EML4-ALK fusion polynucleotide and/or fusionpolypeptide, said method comprising the step of determining whether saidcompound inhibits the expression and/or activity of said EML4-ALK fusionpolypeptide in said cancer. In one preferred embodiment, inhibition ofexpression and/or activity of the EML4-ALK fusion polypeptide or isdetermined using at least one reagent that detects a fusionpolynucleotide and/or fusion polypeptide of the invention. Preferredreagents of the invention have been described above. Compounds suitablefor the inhibition of ALK kinase activity are described in more detailin Section G below.

The compound may, for example, be a kinase inhibitor, such as a smallmolecule or antibody inhibitor. It may be a pan-kinase inhibitor withactivity against several different kinases, or a kinase-specificinhibitor. ALK kinase-inhibiting compounds are discussed in furtherdetail in Section G below. Patient biological samples may be takenbefore and after treatment with the inhibitor and then analyzed, usingmethods described above, for the biological effect of the inhibitor onALK kinase activity, including the phosphorylation of downstreamsubstrate protein. Such a pharmacodynamic assay may be useful indetermining the biologically active dose of the drug that may bepreferable to a maximal tolerable dose. Such information would also beuseful in submissions for drug approval by demonstrating the mechanismof drug action. Identifying compounds with such desired inhibitorycharacteristics is further described in Section G below.

G. Therapeutic Inhibition of Cancers.

In accordance with the present invention, it has now been shown that theprogression of a mammalian solid tumor cancer (NSCLC) in which EML4-ALKfusion protein is expressed may be inhibited, in vivo, by inhibiting theactivity of ALK kinase in such cancer. Similarly as described herein,the activity of a mammalian solid tumor cancer in which TFG-ALK fusionprotein is expressed may be similarly inhibited, in vivo, by inhibitingALK kinase activity in such cancer. ALK activity in cancerscharacterized by expression of a mutant ALK polypeptide may be inhibitedby contacting the cancer (e.g. a tumor) with an ALK kinase-inhibitingtherapeutic, such as a small-molecule kinase inhibitor like WHI-131 orWHI-154. As further described in Example 2 below, growth inhibition ofALK fusion protein-expressing tumors, for example, can be accomplishedby inhibiting the fusion kinase using an exemplary type ofALK-inhibiting therapeutic, siRNA. Accordingly, the invention provides,in part, a method for inhibiting the progression of a cancer thatexpresses EML4-ALK fusion polypeptide or solid tumor that expressesTFG-ALK fusion polypeptide by inhibiting the expression and/or activityof the mutant ALK kinase in the cancer.

An ALK kinase-inhibiting therapeutic may be any composition comprisingat least one compound, biological or chemical, which inhibits, directlyor indirectly, the expression and/or activity of ALK kinase in vivo,including the exemplary classes of compounds described below. Suchcompounds include therapeutics that act directly on ALK kinase itself,or on proteins or molecules that modify the activity of ALK, or that actindirectly by inhibiting the expression of ALK. Such compositions alsoinclude compositions comprising only a single ALK kinase inhibitingcompound, as well as compositions comprising multiple therapeutics(including those against other RTKs), which may also include anon-specific therapeutic agent like a chemotherapeutic agent or generaltranscription inhibitor.

Small-Molecule Inhibitors.

In some preferred embodiments, an ALK-inhibiting therapeutic useful inthe practice of the methods of the invention is a targeted, smallmolecule inhibitor, such as WHI-131 and WHI-154, or their analogues.WHI-131 and WHI-154 are quinazoline-type small molecule targetedinhibitors of ALK, and their properties have been described. See Marzecet al., Lab. Invest. 85(12): 1544-54 (2005). These compounds have beenshown to induce apoptosis and suppress proliferation in lymphoma cells.Other small molecule targeted inhibitors of kinases are well known inthe art. For example, Gleevec® (STI-571, Imatinib), which specificallybinds to and blocks the ATP-binding site of BCR-ABL fusion kinase (aswell as other kinases) thereby preventing phosphorylation and activationof this enzyme, is commercially available and its properties are wellknown. See, e.g., Dewar et al., Blood 105(8): 3127-32 (2005). Othersmall-molecule inhibitors of ALK are presently under development byNovartis, Inc., and Cephalon, Inc.

Small molecule targeted inhibitors are a class of molecules thattypically inhibit the activity of their target enzyme by specifically,and often irreversibly, binding to the catalytic site of the enzyme,and/or binding to an ATP-binding cleft or other binding site within theenzyme that prevents the enzyme from adopting a conformation necessaryfor its activity. Small molecule inhibitors may be rationally designedusing X-ray crystallographic or computer modeling of ALK kinasethree-dimensional structure, or may found by high throughput screeningof compound libraries for inhibition of ALK. Such methods are well knownin the art, and have been described. Specificity of ALK inhibition maybe confirmed, for example, by examining the ability of such compound toinhibit ALK activity, but not other kinase activity, in a panel ofkinases, and/or by examining the inhibition of ALK activity in abiological sample comprising lung carcinoma cells, as described above.Such screening methods are further described below.

Antibody Inhibitors.

ALK kinase-inhibiting therapeutics useful in the methods of theinvention may also be targeted antibodies that specifically bind tocritical catalytic or binding sites or domains required for ALKactivity, and inhibit the kinase by blocking access of ligands,substrates or secondary molecules to ALK, and/or preventing the enzymefrom adopting a conformation necessary for its activity. The production,screening, and therapeutic use of humanized target-specific antibodieshave been well-described. See Merluzzi et al., Adv Clin Path. 4(2):77-85 (2000). Commercial technologies and systems, such as Morphosys,Inc.'s Human Combinatorial Antibody Library (HuCAL®), for thehigh-throughput generation and screening of humanized target-specificinhibiting antibodies are available.

The production of various anti-receptor kinase targeted antibodies andtheir use to inhibit activity of the targeted receptor has beendescribed. See, e.g. U.S. Patent Publication No. 20040202655,“Antibodies to IGF-I Receptor for the Treatment of Cancers,” Oct. 14,2004, Morton et al.; U.S. Patent Publication No. 20040086503, “Humananti-Epidermal Growth Factor Receptor Single-Chain Antibodies,” Apr. 15,2004, Raisch et al.; U.S. Patent Publication No. 20040033543, “Treatmentof Renal Carcinoma Using Antibodies Against the EGFr,” Feb. 19, 2004,Schwab et. al. Standardized methods for producing, and using, receptortyrosine kinase activity-inhibiting antibodies are known in the art.See, e.g., European Patent No. EP1423428, “Antibodies that BlockReceptor Tyrosine Kinase Activation, Methods of Screening for and UsesThereof,” Jun. 2, 2004, Borges et al.

Phage display approaches may also be employed to generate ALK-specificantibody inhibitors, and protocols for bacteriophage libraryconstruction and selection of recombinant antibodies are provided in thewell-known reference text CURRENT PROTOCOLS IN IMMUNOLOGY, Colligan etal. (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section17.1. See also U.S. Pat. No. 6,319,690, Nov. 20, 2001, Little et al.;U.S. Pat. No. 6,300,064, Oct. 9, 2001, Knappik et al.; U.S. Pat. No.5,840,479, Nov. 24, 1998, Little et al.; U.S. Patent Publication No.20030219839, Nov. 27, 2003, Bowdish et al.

A library of antibody fragments displayed on the surface ofbacteriophages may be produced (see, e.g. U.S. Pat. No. 6,300,064, Oct.9, 2001, Knappik et al.) and screened for binding to a soluble dimericform of a receptor protein tyrosine kinase (like ALK). An antibodyfragment that binds to the soluble dimeric form of the RTK used forscreening is identified as a candidate molecule for blockingconstitutive activation of the target RTK in a cell. See European PatentNo. EP1423428, Borges et al., supra.

ALK binding targeted antibodies identified in screening of antibodylibraries as describe above may then be further screened for theirability to block the activity of ALK, both in vitro kinase assay and invivo in cell lines and/or tumors. ALK inhibition may be confirmed, forexample, by examining the ability of such antibody therapeutic toinhibit ALK kinase activity, but not other kinase activity, in a panelof kinases, and/or by examining the inhibition of ALK activity in abiological sample comprising cancer cells, as described above. Methodsfor screening such compounds for ALK kinase inhibition are furtherdescribed above.

Indirect Inhibitors.

ALK-inhibiting compounds useful in the practice of the disclosed methodsmay also be compounds that indirectly inhibit ALK activity by inhibitingthe activity of proteins or molecules other than ALK kinase itself. Suchinhibiting therapeutics may be targeted inhibitors that modulate theactivity of key regulatory kinases that phosphorylate orde-phosphorylate (and hence activate or deactivate) ALK itself, orinterfere with binding of ligands. As with other receptor tyrosinekinases, ALK regulates downstream signaling through a network of adaptorproteins and downstream kinases, including STAT5 and AKT. As a result,induction of cell growth and survival by ALK activity may be inhibitedby targeting these interacting or downstream proteins. Drugs currentlyin development that could be used in this manner include Wartmanin.

ALK kinase activity may also be indirectly inhibited by using a compoundthat inhibits the binding of an activating molecule necessary for ALK toadopt its active conformation. Similarly, for example, the productionand use of anti-PDGF antibodies to down-regulate PDGF receptor tyrosinekinase has been described. See U.S. Patent Publication No. 20030219839,“Anti-PDGF Antibodies and Methods for Producing Engineered Antibodies,”Bowdish et al.

Indirect inhibitors of ALK activity may be rationally designed usingX-ray crystallographic or computer modeling of ALK three dimensionalstructure, or may found by high throughput screening of compoundlibraries for inhibition of key upstream regulatory enzymes and/ornecessary binding molecules, which results in inhibition of ALK kinaseactivity. Such approaches are well known in the art, and have beendescribed. ALK inhibition by such therapeutics may be confirmed, forexample, by examining the ability of the compound to inhibit ALKactivity, but not other kinase activity, in a panel of kinases, and/orby examining the inhibition of ALK activity in a biological samplecomprising cancer cells, e.g. NSCLC cells, as described above. Methodsfor identifying compounds that inhibit a cancer characterized by anEML4-ALK or TFG-ALK fusion polynucleotide and/or fusion polypeptide arefurther described below.

Anti-Sense and/or Transcription Inhibitors.

ALK inhibiting therapeutics may also comprise anti-sense and/ortranscription inhibiting compounds that inhibit ALK kinase activity byblocking transcription of the gene encoding ALK and/or the EML4-ALK orTFG-ALK fusion genes or truncated ALK genes. For example, the inhibitionof various receptor kinases, including VEGFR, EGFR, and IGFR, and FGFR,by antisense therapeutics for the treatment of cancer has beendescribed. See, e.g., U.S. Pat. Nos. 6,734,017; 6,710,174, 6,617,162;6,340,674; 5,783,683; 5,610,288.

Antisense oligonucleotides may be designed, constructed, and employed astherapeutic agents against target genes in accordance with knowntechniques. See, e.g. Cohen, J., Trends in Pharmacol. Sci. 10(11):435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988);Weintraub, H., Sci. AM. pp. 40-46 (1990); Van Der Krol et al.,BioTechniques 6(10): 958-976 (1988); Skorski et al., Proc. Natl. Acad.Sci. USA (1994) 91: 4504-4508. Inhibition of human carcinoma growth invivo using an antisense RNA inhibitor of EGFR has recently beendescribed. See U.S. Patent Publication No. 20040047847, “Inhibition ofHuman Squamous Cell Carcinoma Growth In vivo by Epidermal Growth FactorReceptor Antisense RNA Transcribed from a Pol III Promoter,” Mar. 11,2004, He et al. Similarly, an ALK-inhibiting therapeutic comprising atleast one antisense oligonucleotide against a mammalian ALK gene (seeFIG. 4 (SEQ ID NO: 6)) or EML4-ALK or TFG-ALK fusion polynucleotide ortruncated ALK polynucleotide (see FIGS. 2A-C(SEQ ID NOs: 2, 19, and 21))may be prepared according to methods described above. Pharmaceuticalcompositions comprising ALK-inhibiting antisense compounds may beprepared and administered as further described below.

Small Interfering RNA.

Small interfering RNA molecule (siRNA) compositions, which inhibittranslation, and hence activity, of ALK through the process of RNAinterference, may also be desirably employed in the methods of theinvention. RNA interference, and the selective silencing of targetprotein expression by introduction of exogenous small double-strandedRNA molecules comprising sequence complimentary to mRNA encoding thetarget protein, has been well described. See, e.g. U.S. PatentPublication No. 20040038921, “Composition and Method for InhibitingExpression of a Target Gene,” Feb. 26, 2004, Kreutzer et al.; U.S.Patent Publication No. 20020086356, “RNA Sequence-Specific Mediators ofRNA Interference,” Jun. 12, 2003, Tuschl et al.; U.S. Patent Publication20040229266, “RNA Interference Mediating Small RNA Molecules,” Nov. 18,2004, Tuschl et. al.

For example, as presently shown (see Example 2), siRNA-mediatedsilencing of expression of the EML4-ALK fusion protein in a human NSCLCcell line expressing the fusion protein selectively inhibited theprogression of the disease in those cells, but not in control cells thatdo not express the mutant ALK protein.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNAse III Dicer processes dsRNA intosmall interfering RNAs (siRNA) of approximately 22 nucleotides, whichserve as guide sequences to induce target-specific mRNA cleavage by anRNA-induced silencing complex RISC (see Hammond et al., Nature (2000)404: 293-296). RNAi involves a catalytic-type reaction whereby newsiRNAs are generated through successive cleavage of longer dsRNA. Thus,unlike antisense, RNAi degrades target RNA in a non-stoichiometricmanner. When administered to a cell or organism, exogenous dsRNA hasbeen shown to direct the sequence-specific degradation of endogenousmessenger RNA (mRNA) through RNAi.

A wide variety of target-specific siRNA products, including vectors andsystems for their expression and use in mammalian cells, are nowcommercially available. See, e.g. Promega, Inc. (promega.com);Dharmacon, Inc. (dharmacon.com). Detailed technical manuals on thedesign, construction, and use of dsRNA for RNAi are available. See, e.g.Dharmacon's “RNAi Technical Reference & Application Guide”; Promega's“RNAi: A Guide to Gene Silencing.” ALK-inhibiting siRNA products arealso commercially available, and may be suitably employed in the methodof the invention. See, e.g. Dharmacon, Inc., Lafayette, Colo. (Cat Nos.M-003162-03, MU-003162-03, D-003162-07 thru -10 (siGENOME™SMARTselection and SMARTpool® siRNAs).

It has recently been established that small dsRNA less than 49nucleotides in length, and preferably 19-25 nucleotides, comprising atleast one sequence that is substantially identical to part of a targetmRNA sequence, and which dsRNA optimally has at least one overhang of1-4 nucleotides at an end, are most effective in mediating RNAi inmammals. See U.S. Patent Publication No. 20040038921, Kreutzer et al.,supra; U.S. Patent Publication No. 20040229266, Tuschl et al., supra.The construction of such dsRNA, and their use in pharmaceuticalpreparations to silence expression of a target protein, in vivo, aredescribed in detail in such publications.

If the sequence of the gene to be targeted in a mammal is known, 21-23nt RNAs, for example, can be produced and tested for their ability tomediate RNAi in a mammalian cell, such as a human or other primate cell.Those 21-23 nt RNA molecules shown to mediate RNAi can be tested, ifdesired, in an appropriate animal model to further assess their in vivoeffectiveness. Target sites that are known, for example target sitesdetermined to be effective target sites based on studies with othernucleic acid molecules, for example ribozymes or antisense, or thosetargets known to be associated with a disease or condition such as thosesites containing mutations or deletions, can be used to design siRNAmolecules targeting those sites as well.

Alternatively, the sequences of effective dsRNA can be rationallydesigned/predicted screening the target mRNA of interest for targetsites, for example by using a computer folding algorithm. The targetsequence can be parsed in silico into a list of all fragments orsubsequences of a particular length, for example 23 nucleotidefragments, using a custom Perl script or commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package.

Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siRNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. See, e.g., U.S. Patent Publication No. 20030170891, Sep. 11,2003, McSwiggen J. An algorithm for identifying and selecting RNAitarget sites has also recently been described. See U.S. PatentPublication No. 20040236517, “Selection of Target Sites for AntisenseAttack of RNA,” Nov. 25, 2004, Drlica et al.

Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods(Graham et al. (1973) Virol. 52: 456; McCutchan et al., (1968), J. Natl.Cancer Inst. 41: 351; Chu et al. (1987), Nucl. Acids Res. 15: 1311;Fraley et al. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), Cell22: 479). DNA may also be introduced into cells using cationic liposomes(Feigner et al. (1987), Proc. Natl. Acad. Sci USA 84:7413). Commerciallyavailable cationic lipid formulations include Tfx 50 (Promega) orLipofectamin 200 (Life Technologies). Alternatively, viral vectors maybe employed to deliver dsRNA to a cell and mediate RNAi. See U.S PatentPublication No. 20040023390, “siRNA-mediated Gene Silencing with ViralVectors,” Feb. 4, 2004, Davidson et al.

Transfection and vector/expression systems for RNAi in mammalian cellsare commercially available and have been well described. See, e.g.Dharmacon, Inc., DharmaFECT™ system; Promega, Inc., siSTRIKE™ U6 Hairpinsystem; see also Gou et al. (2003) FEBS. 548, 113-118; Sui, G. et al. ADNA vector-based RNAi technology to suppress gene expression inmammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al.(2002) Proc. Natl. Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002)Nature Biotechnology 19, 505-508; McManus et al. (2002) RNA 8, 842-850.

siRNA interference in a mammal using prepared dsRNA molecules may thenbe effected by administering a pharmaceutical preparation comprising thedsRNA to the mammal. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene. dsRNA cantypically be administered at a dosage of less than 5 mg dsRNA perkilogram body weight per day, and is sufficient to inhibit or completelysuppress expression of the target gene. In general a suitable dose ofdsRNA will be in the range of 0.01 to 2.5 milligrams per kilogram bodyweight of the recipient per day, preferably in the range of 0.1 to 200micrograms per kilogram body weight per day, more preferably in therange of 0.1 to 100 micrograms per kilogram body weight per day, evenmore preferably in the range of 1.0 to 50 micrograms per kilogram bodyweight per day, and most preferably in the range of 1.0 to 25 microgramsper kilogram body weight per day. A pharmaceutical compositioncomprising the dsRNA is administered once daily, or in multiplesub-doses, for example, using sustained release formulations well knownin the art. The preparation and administration of such pharmaceuticalcompositions may be carried out accordingly to standard techniques, asfurther described below.

Such dsRNA may then be used to inhibit ALK expression and activity in acancer, by preparing a pharmaceutical preparation comprising atherapeutically-effective amount of such dsRNA, as described above, andadministering the preparation to a human subject having a cancerexpressing EML4-ALK or TFG-ALK fusion protein or truncated active ALKkinase, for example, via direct injection to the tumor. The similarinhibition of other receptor tyrosine kinases, such as VEGFR and EGFRusing siRNA inhibitors has recently been described. See U.S. PatentPublication No. 20040209832, Oct. 21, 2004, McSwiggen et al.; U.S.Patent Publication No. 20030170891, Sep. 11, 2003, McSwiggen; U.S.Patent Publication No. 20040175703, Sep. 9, 2004, Kreutzer et al.

Therapeutic Compositions: Administration.

ALK kinase-inhibiting therapeutic compositions useful in the practice ofthe methods of the invention may be administered to a mammal by anymeans known in the art including, but not limited to oral or peritonealroutes, including intravenous, intramuscular, intraperitoneal,subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical(including buccal and sublingual) administration.

For oral administration, an ALK-inhibiting therapeutic will generally beprovided in the form of tablets or capsules, as a powder or granules, oras an aqueous solution or suspension. Tablets for oral use may includethe active ingredients mixed with pharmaceutically acceptable excipientssuch as inert diluents, disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloring agentsand preservatives. Suitable inert diluents include sodium and calciumcarbonate, sodium and calcium phosphate, and lactose, while corn starchand alginic acid are suitable disintegrating agents. Binding agents mayinclude starch and gelatin, while the lubricating agent, if present,will generally be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate, to delay absorption in the gastrointestinaltract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil. For intramuscular,intraperitoneal, subcutaneous and intravenous use, the pharmaceuticalcompositions of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. The carrier may consist exclusively of anaqueous buffer (“exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of theALK-inhibiting therapeutic). Such substances include, for example,micellar structures, such as liposomes or capsids, as described below.Aqueous suspensions may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

ALK kinase-inhibiting therapeutic compositions may also includeencapsulated formulations to protect the therapeutic (e.g. a dsRNAcompound) against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075. An encapsulatedformulation may comprise a viral coat protein. The viral coat proteinmay be derived from or associated with a virus, such as a polyoma virus,or it may be partially or entirely artificial. For example, the coatprotein may be a Virus Protein 1 and/or Virus Protein 2 of the polyomavirus, or a derivative thereof.

ALK-inhibiting compositions can also comprise a delivery vehicle,including liposomes, for administration to a subject, carriers anddiluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations. For example, methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDETHERAPEUTICS, ed. Akbtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192. Beigelmanet al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595further describe the general methods for delivery of nucleic acidmolecules. These protocols can be utilized for the delivery of virtuallyany nucleic acid molecule.

ALK-inhibiting therapeutics can be administered to a mammalian tumor bya variety of methods known to those of skill in the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (O'Hare and Normand, International PCT PublicationNo. WO 00/53722). Alternatively, the therapeutic/vehicle combination islocally delivered by direct injection or by use of an infusion pump.Direct injection of the composition, whether subcutaneous,intramuscular, or intradermal, can take place using standard needle andsyringe methodologies, or by needle-free technologies such as thosedescribed in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 andBarry et al., International PCT Publication No. WO 99/31262.

Pharmaceutically acceptable formulations of ALK kinase-inhibitorytherapeutics include salts of the above-described compounds, e.g., acidaddition salts, for example, salts of hydrochloric, hydrobromic, aceticacid, and benzene sulfonic acid. A pharmacological composition orformulation refers to a composition or formulation in a form suitablefor administration, e.g., systemic administration, into a cell orpatient, including for example a human. Suitable forms, in part, dependupon the use or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell. For example, pharmacological compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the composition or formulation from exerting its effect.

Administration routes that lead to systemic absorption (i.e. systemicabsorption or accumulation of drugs in the blood stream followed bydistribution throughout the entire body), are desirable and include,without limitation: intravenous, subcutaneous, intraperitoneal,inhalation, oral, intrapulmonary and intramuscular. Each of theseadministration routes exposes the ALK-inhibiting therapeutic to anaccessible diseased tissue or tumor. The rate of entry of a drug intothe circulation has been shown to be a function of molecular weight orsize. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Nonlimiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich etal, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.);and loaded nanoparticles, such as those made of polybutylcyanoacrylate,which can deliver drugs across the blood brain barrier and can alterneuronal uptake mechanisms (Prog Neuro-psychopharmacol Biol Psychiatry,23, 941-949, 1999). Other non-limiting examples of delivery strategiesfor the ALK-inhibiting compounds useful in the method of the inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

Therapeutic compositions comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes) may also be suitablyemployed in the methods of the invention. These formulations offer amethod for increasing the accumulation of drugs in target tissues. Thisclass of drug carriers resists opsonization and elimination by themononuclear phagocytic system (MPS or RES), thereby enabling longerblood circulation times and enhanced tissue exposure for theencapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes havebeen shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; Choi et al., International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

Therapeutic compositions may include a pharmaceutically effective amountof the desired compounds in a pharmaceutically acceptable carrier ordiluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inREMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A. R. Gennaro,Ed. 1985). For example, preservatives, stabilizers, dyes and flavoringagents can be provided. These include sodium benzoate, sorbic acid andesters of p-hydroxybenzoic acid. In addition, antioxidants andsuspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient. It is understood that the specific dose level for anyparticular patient depends upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

An ALK-inhibiting therapeutic useful in the practice of the inventionmay comprise a single compound as described above, or a combination ofmultiple compounds, whether in the same class of inhibitor (i.e.antibody inhibitor), or in different classes (i.e antibody inhibitorsand small-molecule inhibitors). Such combination of compounds mayincrease the overall therapeutic effect in inhibiting the progression ofa fusion protein-expressing cancer. For example, the therapeuticcomposition may a small molecule inhibitor, such as WHI-131 and/orWHI-154 alone, or in combination with other inhibitors targeting ALKactivity and/or other small molecule inhibitors. The therapeuticcomposition may also comprise one or more non-specific chemotherapeuticagent in addition to one or more targeted inhibitors. Such combinationshave recently been shown to provide a synergistic tumor killing effectin many cancers. The effectiveness of such combinations in inhibitingALK activity and tumor growth in vivo can be assessed as describedbelow.

Identification of Mutant ALK Kinase-Inhibiting Compounds.

The invention also provides, in part, a method for determining whether acompound inhibits the progression of a cancer characterized by anEML4-ALK or TFG-ALK fusion polynucleotide and/or fusion polypeptide, bydetermining whether the compound inhibits the activity of EML4-ALK orTFG-ALK fusion polypeptide or truncated ALK kinase polypeptide in thecancer. In some preferred embodiments, inhibition of activity of ALK isdetermined by examining a biological sample comprising cells from bonemarrow, blood, pleural effusion, or a tumor. In another preferredembodiment, inhibition of activity of ALK is determined using at leastone mutant ALK polynucleotide or polypeptide-specific reagent of theinvention.

The tested compound may be any type of therapeutic or composition asdescribed above. Methods for assessing the efficacy of a compound, bothin vitro and in vivo, are well established and known in the art. Forexample, a composition may be tested for ability to inhibit ALK in vitrousing a cell or cell extract in which ALK is activated. A panel ofcompounds may be employed to test the specificity of the compound forALK (as opposed to other targets, such as EGFR or PDGFR).

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity to aprotein of interest, as described in published PCT applicationWO84/03564. In this method, as applied to mutant ALK polypeptides, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with mutant ALK polypeptide, or fragments thereof,and washed. Bound mutant polypeptide (e.g. EML4-ALK fusion polypeptide)is then detected by methods well known in the art. Purified mutant ALKpolypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

A compound found to be an effective inhibitor of ALK activity in vitromay then be examined for its ability to inhibit the progression of acancer expressing EML4-ALK or TFG-ALK fusion polypeptide and/ortruncated ALK kinase polypeptide, in vivo, using, for example, mammalianxenografts harboring human tumors, such as NSCLC. In this way, theeffects of the drug may be observed in a biological setting most closelyresembling a patient. The drug's ability to alter signaling in thecancerous cells or surrounding stromal cells may be determined byanalysis with phosphorylation-specific antibodies. The drug'seffectiveness in inducing cell death or inhibition of cell proliferationmay also be observed by analysis with apoptosis specific markers such ascleaved caspase 3 and cleaved PARP. Similarly, mammalian bone marrowtransplants (e.g. mice) harboring human leukemias that are driven by themutant ALK protein may be employed. In this procedure, bone marrow cellsknown to be driven by mutant ALK kinase are transplanted in the mouse.The growth of the cancerous cells may be monitored. The mouse may thenbe treated with the drug, and the effect of the drug treatment on cancerphenotype or progression be externally observed. The mouse is thensacrificed and the transplanted bone marrow removed for analysis by,etc., IHC and Western blot.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The teachings of all references cited above and below are herebyincorporated herein by reference. The following Examples are providedonly to further illustrate the invention, and are not intended to limitits scope, except as provided in the claims appended hereto. The presentinvention encompasses modifications and variations of the methods taughtherein which would be obvious to one of ordinary skill in the art.

Example 1 Identification of ALK Kinase Activity in Solid Tumors byGlobal Phosphopeptide Profiling A. Profiling of Human NSCLC Cell Lines.

The global phosphorylation profile of kinase activation in 22 humanNSCLC cell lines, including H2228, were examined using a recentlydescribed and powerful technique for the isolation and massspectrometric characterization of modified peptides from complexmixtures (the “IAP” technique, see Rush et al., supra). The IAPtechnique was performed using a phosphotyrosine-specific antibody (CELLSIGNALING TECHNOLOGY, INC., Beverly, Mass., 2003/04 Cat. #9411) toisolate, and subsequently characterize, phosphotyrosine-containingpeptides from extracts of the NSCLC cell lines.

Specifically, the IAP approach was employed go facilitate theidentification of tyrosine kinases responsible for proteinphosphorylation in each of the NSCLC cell lines. In particular, atypicalor unusual kinase activity was considered.

Cell Culture.

All cell culture reagents were purchased from Invitrogen, Inc. A totalof 41 human NSCLC cell lines were examined. Human NSCLC cell lines,H520, H838, H1437, H1563, H1568, H1792, H1944, H2170, H2172, H2228,H2347, A549, H441, H1703, H1373, and H358, were obtained from AmericanType Culture Collection, and cultured in RPMI 1640 medium with 10% FBSand adjusted to contain 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate,penicillin/streptomycin. An additional six human NSCLC cell lines,HCC78, Cal-12T, HCC366, HCC15, HCC44, and LOU-NH91, were purchased fromDSMZ, and cultured in RPMI 1640 containing 10% FBS andpenicillin/streptomycin. Cells were maintained in a 5% CO2 incubator at37° C.

For the immunoaffinity precipitation and immunoblot experiments, cellswere grown to 80% confluence and then starved in RPMI medium without FBSovernight before harvesting.

Phosphopeptide Immunoprecipitation.

100 million cells were lysed in urea lysis buffer (20 mM Hepes, pH 8.0,9 M Urea, I mM sodium vanadate, 2.5 mM sodium pyrophosphate, 1 mMbeta-glycerophosphate). The lysate was sonicated and cleared bycentrifugation. Cleared lysate was reduced by DTT and alkylated withiodoacetamide, as described previously (see Rush et al., Nat.Biotechnol. 23(1): 94-101 (2005)). Samples were then diluted 4 timeswith 20 mM Hepes to reduce Urea concentration to 2M, and digested bytrypsin overnight at room temperature with gentle shaking.

Digested peptides were crudely purified with Sep-Pak C18 columns, aspreviously described (see Rush et al., supra). Elute was lyophilized anddried peptides were dissolved in 1.4 ml of MOPS IP buffer (50 mMMOPS/NaOH pH 7.2, 10 mM Na2PO4, 50 mM NaCl) and insoluble materialremoved by centrifugation. Immunoprecipitation was carried at 4° C. forovernight with 160 μg of Phospho-Tyrosine 100 antibody (Cell SignalingTechnology) coupled to protein G agarose beads (Roche). The beads werethen washed 3 times with 1 ml MOPS IP buffer and twice with 1 ml HPLCgrade dH2O in the cold. Phosphopeptides were eluted from beads with 60μl 0.1% TFA followed by a second elution with 40 μl 0.1% TFA and thefractions were pooled. The eluted peptides were concentrated using aZipTip column (Millipore), and analyzed with LC-MS/MS. Mass spectra werecollected with an LTQ ion trap mass spectrometer (ThermoFinnigan).

Analysis by LC-MS/MS Mass Spectrometry.

Peptides in the IP eluate (100 μl) were concentrated and separated fromeluted antibody using Stop and Go extraction tips (StageTips) (seeRappsilber et al., Anal. Chem., 75(3): 663-70 (2003)). Peptides wereeluted from the microcolumns with 1 μl of 60% MeCN, 0.1% TFA into 7.6 μlof 0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA).

Each phosphopeptide sample was LC-MS analyzed in duplicate. A fusedsilica microcapillary column (125 μm×18 cm) was packed with C18reverse-phase resin (Magic C18AQ, 5 μm particles, 200 Å pore size,Michrom Bioresources, Auburn, Calif.). Samples (4 μL) were loaded ontothis column with an autosampler (LC Packings Famos, San Francisco,Calif.) and eluted into the mass spectrometer by a 55-min lineargradient of 7 to 30% acetonitrile in 0.1% formic acid. The gradient wasdelivered at approximately abc nl/min using a binary HPLC pump (Agilent1100, Palo Alto, Calif.) with an in-line flow splitter. Eluting peptideions were mass analyzed with a hybrid linear ion trap-7 Tesla ioncyclotron resonance Fourier transform instrument (LTQ-FT, ThermoFinnigan, San Jose, Calif.).

A top-seven method was employed, whereby 7 data-dependent MS/MS scans inthe linear ion trap were collected based on measurements made during theprevious MS survey scan in the ICR cell, with the linear ion trap andthe Fourier transform instrument operating concurrently. MS scans wereperformed at 375-1800 m/z with an automatic gain control (AGC) target of8×106 and a mass resolution of 105. For MS/MS the AGC was 8×106, thedynamic exclusion time was 25 s, and singly-charged ions were rejectedby charge-state screening.

Database Analysis & Assignments.

Peptide sequences were assigned to MS/MS spectra using TurboSequestsoftware (v.27, rev. 12) (ThermoFinnigan) and a compositeforward/reverse IPI human protein database. Search parameters were:trypsin as protease; 1.08 Da precursor mass tolerance; staticmodification on cysteine (+57.02146, carboxamidomethylation); anddynamic modifications on serine, threonine and tyrosine (+79.96633 Da,phosphorylation), lysine (+8.01420, 13C615N2), arginine (+6.02013, 13C6)and methionine (+15.99491, oxidation). A target/decoy database approachwas used to establish appropriate score-filtering criteria such that theestimated false-positive assignment rate was <1%. In addition toexceeding charge-dependent XCorr thresholds (z=1, XCorr≥1.5, for z=2,XCorr≥2.2, for z=3, XCorr≥3.3), assignments were required to containphosphotyrosine, to have a mass accuracy of −5 to +25 ppm, and tocontain either all-light or all-heavy lysine/arginine residues.

Assignments passing these criteria were further evaluated using a customquantification program, Vista (Bakalarski et al., manuscript inpreparation) to calculate peak areas and ultimately a relative abundancebetween heavy and light forms of each peptide. Identified peptides withsignal-to-noise in the MS scan below 15 were not considered forquantification. For those peptides found only in one of the conditionsthe signal-to-noise ratio was used instead.

Searches were done against the NCBI human database released on Aug. 24,2004 containing 27,175 proteins allowing oxidized methionine (M+16) andphosphorylation (Y+80) as dynamic modifications. All spectra supportingthe final list of assigned sequences (not shown here) were reviewed byat least three scientists to establish their credibility.

The foregoing IAP analysis identified over 2000 non-redundantphosphotyrosine-containing peptides, over 1,500 phosphotyrosine sites,and more than 1,000 tyrosine phosphorylated proteins, the majority ofwhich are novel, from the cell lines examined (data not shown). Receptortyrosine kinases known to be involved in NSCLC signaling were observedto be tyrosine phosphorylated in many cell lines, such as EGFR, Her2,Her3, EphA2 and Met. High levels of EGFR phosphopeptides were observedin several cell lines including HCC827 and H3255, two cell lines knownto express amplified levels of genetically activated forms of EGFRconfirming the method identifies receptor tyrosine kinases known to beactive in NSCLC cell lines.

Three cell lines expressed receptor tyrosine kinases not observed inother NSCLC cell lines. Large amounts of tyrosine phosphorylatedpeptides from Ros, ALK, and PDGFR alpha were observed in HCC78, H2228,and H1703 cell lines respectively. The NSCLC cell line H2228, whichhighly expresses ALK, was selected for further examination.

B. Profiling of Human NSCLC Tumor Samples.

The IAP technique, substantially as described in Part A above, wassubsequently applied to examine global phospho-profiles of a panel of154 human tumor samples from NSCLC patients. Tissues were obtained fromthe Second Xiangya Hospital, China.

Frozen tissue samples were cut into small pieces, homogenized in lysisbuffer (20 mM HEPES pH 8.0, 9 M Urea, 1 mN sodium vanadate, supplementedwith 2.5 mM sodium pyrophosphate, 1 mM b-glycerol-phosphate, 1 ml lysisbuffer for 100 mg of frozen tissue) using a polytron for 2 times of 20sec. each time. Homogenate was then briefly sonicated. Cleared lysatewas reduced by DTT and alkylated with iodoacetamide, as describedpreviously (see Rush et al., Nat. Biotechnol. 23(1): 94-101 (2005)).Samples were then diluted 4 times with 20 mM Hepes to reduce Ureaconcentration to 2M, and digested by trypsin overnight at roomtemperature with gentle shaking.

Digested peptides were crudely purified with Sep-Pak C18 columns, aspreviously described (see Rush et al., supra). Elute was lyophilized anddried peptides were dissolved in 1.4 ml of MOPS IP buffer (50 mMMOPS/NaOH pH 7.2, 10 mM Na2PO4, 50 mM NaCl) and insoluble materialremoved by centrifugation. Immunoprecipitation was carried at 4° C. forovernight with 160 μg of Phospho-Tyrosine 100 antibody (Cell SignalingTechnology) coupled to protein G agarose beads (Roche). The beads werethen washed 3 times with 1 ml MOPS IP buffer and twice with 1 ml HPLCgrade dH2O in the cold. Phosphopeptides were eluted from beads with 60μl 0.1% TFA followed by a second elution with 40 μl 0.1% TFA and thefractions were pooled. The eluted peptides were concentrated using aZipTip column (Millipore), and analyzed with LC-MS/MS. Mass spectra werecollected with an LTQ ion trap mass spectrometer (ThermoFinnigan).

Phosphopeptide immunoprecipitation, followed by LC-MS/MS spectrometryanalysis was then carried out as described above in Part A. Databasesearching and sequence assignments were made substantially as describedabove in Part A, but using the NCBI human database released on Aug. 24,2004 containing 27,970 proteins.

The foregoing IAP analysis identified over 2000 non-redundantphosphotyrosine-containing peptides, over 1,500 phosphotyrosine sites,and more than 1,000 tyrosine phosphorylated proteins from the humantumor samples examined (data not shown). Receptor tyrosine kinases knownto be involved in NSCLC signaling were again observed to be tyrosinephosphorylated in many tumors, such as EGFR, Her2, Her3, EphA2 and Met.High levels of EGFR phosphopeptides were again observed in several tumorsamples confirming that the method identifies receptor tyrosine kinasesknown to be active in NSCLC cell lines.

Five patient samples expressed receptor tyrosine kinases not observed inother NSCLC cell lines and tumors. Large amounts oftyrosine-phosphorylated peptides from ALK were observed in patientsCS010/11, CS045, and CS110. These three tumors, which highly expressALK, were selected for further examination.

Example 2 Isolation & Sequencing of Three ALK Fusion Genes A. Sequencingin Human NSCLC Cell Line.

Given the high phosphorylation level of ALK kinase detected in the NSCLCcell line H2228, 5′ rapid amplification of cDNA ends on the sequenceencoding the kinase domain of ALK was conducted in order to determinewhether a chimeric ALK transcript was present.

Rapid Amplification of Complementary DNA Ends

RNeasy Mini Kit (Qiagen) was used to extract RNA from the H2228 cellline. DNA was extracted with the use of DNeasy Tissue Kit (Qiagen).Rapid amplification of cDNA ends was performed with the use of 5′ RACEsystem (Invitrogen) with primers ALK-GSP1 for cDNA synthesis andALK-GSP2 and ALK-GSP3 for a nested PCR reaction.

5′ RACE

FIG. 5 (panel A) shows the detection of the EML4-ALK fusion gene (shortvariant) by 5′RACE and the detection of the PCR amplification productafter 2 rounds. The PCR product was purified with PCR purification kit(Qiagen) and sequenced using ALK-GSP3 an ABI 3130 capillary automaticDNA sequencer (Applied Biosystems). Sequence analysis of the resultantproduct revealed that the kinase domain and C-terminal of ALK was fusedto the EML-4 gene N-terminus (see FIG. 1, panel B). The EML4-ALK fusiongene (short variant) was in-frame and fused the first 233 amino acids ofEML-4 to the last 562 amino acids of ALK (see FIG. 1, panel B). EML-4and ALK genes are both located on chromosome 2, thus the fusion gene wascreated by gene deletion between these two loci.

The following primers were used:

(SEQ ID NO: 9) ALK-GSP1: 5′-GCAGTAGTTGGGGTTGTAGTC (SEQ ID NO: 10)ALK-GSP2: 5′-GCGGAGCTTGCTCAGCTTGT (SEQ ID NO: 11) ALK-GSP3:5′-TGCAGCTCCTGGTGCTTCC

PCR Assay

RT-PCR analysis was performed to confirm the N-terminus of EML-4 isintact in the fusion protein (see FIG. 6 (panel B)). First-strand cDNAwas synthesized from 2.5 mg of total RNA with the use of SuperScript™III first-strand synthesis system (Invitrogen) with oligo (dT)₂₀. Then,the EML4-ALK fusion gene was amplified with the use of primer pairsEML-Atg and ALK-GSP3. The reciprocal fusion was detected with the use ofprimer pairs EML-4-43 and ALK-GSP3 and EML-4-94 and ALK-GSP3 andEML4-202 and ALK-GSP3. For genomic PCR, amplification of the fusion genewas performed with the use of Platinum Taq DNA polymerase high fidelity(Invitrogen) with primer pairs EML-4-atg and ALK-tga.

The following primers were used:

(SEQ ID NO: 12) ALK-GSP3: 5′-TGCAGCTCCTGGTGCTTCC (SEQ ID NO: 13)EML4-Atg: 5′-CGCAAGATGGACGGTTTGGC (SEQ ID NO: 14) EML4-43:5′-TGTTCAAGATCGCCTGTCAGCTCT (SEQ ID NO: 15) EML4-94:5′-TGAAATCACTGTGCTAAAGGCGGC (SEQ ID NO: 16) EML4-202:5′-AAGCCCTCGAGCAGTTATTCCCAT (SEQ ID NO: 17) ALK-Tga:5′-GAATTCCGCCGAGCTCAGGGCCCAG

Of note, in the EML4-ALK fusion (short variant), the ALK moiety is fusedto the EML-4 moiety at precisely the same point in ALK has been observedin other ALK fusions, such as the NPM-ALK fusion occurring in ALCL. Thekinase domain of ALK in the H2228 cell line was further sequenced fromgenomic DNA and found to be wild type. Hence, the deletion mutationdiscovered in H2228 does not affect the ALK kinase domain. Further, wildtype EML-4 is tyrosine phosphorylated only at a site that is not presentin the EML4-ALK fusion protein (short variant), suggesting that theN-terminal coiled coil domain that is conserved in the fusion protein(see FIG. 1A) may function to dimerize and activate ALK, as well as topromote interaction with wild type ALK.

B. Sequencing in Human NSCLC Cell Line.

Similarly, given the high phosphorylation level of ALK kinase detectedin the human NSCLC tumor samples from patients CS010/11, CS045, andCS110, 5′ rapid amplification of cDNA ends on the sequence encoding thekinase domain of ALK was conducted in order to determine whether achimeric ALK transcript was present in these tumors.

Rapid amplification of complementary DNA ends and 5′ RACE was carriedout, substantially as described above in Part A, with primers ALK-GSP1for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for a nested PCR reaction.

FIG. 5 (panel C) shows the detection of the EML4-ALK fusion genes (bothshort and long variants) by 5′RACE in two patient samples, and thedetection of the TFG-ALK fusion gene in one patient, and the detectionof the PCR amplification product after 2 rounds. The PCR products werepurified and sequenced substantially as described above in Part A.Sequence analysis of the resultant products revealed that the kinasedomain and C-terminal of ALK were fused to the EML-4 gene N-terminus intwo different variants (see FIGS. 1A-1B, panel B). The EML4-ALK fusiongenes were in-frame and fused the first 233 amino acids (short variant)or first 495 amino acids (long variant) of EML-4 to the last 562 aminoacids of ALK (see FIGS. 1A-1B, panel B). EML-4 and ALK genes are bothlocated on chromosome 2, thus the fusion gene was created by genedeletion between these two loci. The observation of the fusion gene(short variant) in patient CS045 confirmed the finding of this mutantgene in the human cell line H2228.

The TFG-ALK fusion gene was also in-frame and fused the first 138 aminoacids of TFG to the last 562 amino acids of ALK (see FIG. 1C, panel B).TFG and ALK genes are located on different chromosomes (chromosomes 6and 2, respectively), thus the fusion gene was created by genetranslocation between these two loci. Interestingly, the fusion of TPGto ALK occurred at exactly the same point in ALK as observed for thefusion of the two EML4-ALK variants, indicating that truncation of ALKin solid tumors at this point may be a common occurrence.

The same primers were used as described in Part A above. RT-PCR analysiswas performed, substantially as described in Part A above, to confirmthe N-terminus of EML-4 and TFG are intact in the fusion proteins (seeFIG. 6 (panel B)). Primer pairs for EML-4 and ALK were as described inPart A above. The following primer pair was used for TFG:

(SEQ ID NO: 28) TFG-F1: 5′-TTTGTTAATGGCCAGCCAAGACCC-3

Of note, in both EML4-ALK fusion variants, the ALK moiety is fused tothe EML-4 moiety at precisely the same point in ALK has been observed inother ALK fusions, such as the NPM-ALK fusion occurring in ALCL.Further, wild type EML-4 is tyrosine phosphorylated only at a site thatis not present in the EML4-ALK fusion proteins, suggesting that theN-terminal coiled coil domain that is conserved in the fusion proteins(see FIGS. 1A-1B) may function to dimerize and activate ALK, as well asto promote interaction with wild type ALK. Also of note, the fusion ofthe TG moiety to ALK also occurs at precisely the same point in ALK, andindeed the fusion of TFG to ALK at this point has been described inhuman lymphoma (see Hernandez et al. (2002), supra.), but has notpreviously been described in human solid tumors, such as NSCLC.

Example 3 Growth Inhibition of ALK Fusion-Expressing Mammalian SolidTumors Using siRNA

In order to confirm that the truncated/fusion forms of ALK are drivingcell growth and survival in NSCLC cell line H2228 as well as NSCLC tumorsamples from patients CS010/11, CS045, and CS110, the ability of siRNA(against ALK) to inhibit growth of these cells and tumors may beexamined.

ALK SMARTpool siRNA duplexes (proprietary target sequences—data notshown) may be purchased, for example, from Dharmacon Research, Inc.(Lafayette, Colo.). A non-specific SMARTpool siRNA is used as a control.Cells are transfected with the siRNA via electroporation. Briefly, 2×10⁷cells (H2228) are pulsed once (20 ms; 275V, K562 20 ms; 285V) using asquare-wave electroporator (BTX Genetronics, San Diego, Calif.),incubated at room temperature for 30 minutes and transferred to T150flasks with 30 ml RPMI-1640/10% FBS.

The number of viable cells is determined with the CellTiter 96AQ_(ueous) One solution cell proliferation assay (Promega). IC₅₀ iscalculated with the use of OriginPro 6.1 software (OriginLab). Thepercentage of apoptotic cells at 48 hours may be determined by flowcytometric analysis of Cleaved-Caspase-3 (Cell Signaling Technology).

Immunoblot analysis will reveal that the expression of ALK isspecifically and significantly reduced at 72 hours followingtransfection of the siRNA into H2228 cells or tumor cells from patientsCS010/11, CS045, and CS110. Down regulation of ALK is expected to resultin strong inhibition of cell growth. Treatment with ALK siRNA is alsoexpected to result in increased apoptosis of these solid tumor cells.These results will further indicate that the mutant/fused ALK kinases inthe H2228 cell line and patient tumors are driving the proliferation andgrowth of these NSCLC cells, and that such growth and proliferation maybe inhibited by using siRNA to inhibit ALK kinase expression andactivity.

Example 4 Growth Inhibition of ALK Fusion-Expressing Mammalian SolidTumors Using WI-131 and/or WI-154

To further confirm that the mutant ALK fusion proteins are driving thegrowth and viability of the NSCLC cell line H2228 and NSCLC tumor cellsfrom patients CS010/11, CS045, and CS110, the cells may be treated witha targeted inhibitor of ALK kinase, such as WI-131 and/or WI-154. WI-131and W-154 are quinazoline-type small molecule inhibitors of ALK kinase,and their activity against the NPM-ALK fusion protein in T-cell lymphomahas been described. See Marzec et al., supra.

Briefly, NSCLC cells are cultured, and a cell growth inhibition assay isperformed with CellTiter 96 AQueous One Solution Cell ProliferationAssay (Promega) according to manufacturer's suggestion. Briefly, 1000 to5000 cells are seeded onto flat-bottomed 96-well plates and grown incomplete medium with 10% FBS. After 24 hours, the cell medium is changedto 100 μl complete growth medium with 10% FBS containing variousconcentrations of the drug, and the cells are incubated for anadditional 72 hours. Each drug concentration is applied to triplicatewell of cells. At the end of the incubation, 20 μl of CellTiter 96AQ_(ueous) One solution is added to each well, and the plate wasincubated for 1-4 hours. Absorbance is read at 490 nm using a TitanMultiskan Ascent microplate reader (Titertek Instrument). Growthinhibition may be expressed as mean±SD value of percentage of absorbancereading from treated cells versus untreated cells. The assay is repeatedat least three times.

Such analysis is expected to confirm that the ALK fusion proteins(EML4-ALK (short and long variants), TFG-ALK) are driving growth andsurvival of a subset of human NSCLC tumors in which these mutantproteins are expressed, and that such cells may be inhibited byinhibiting the activity of the fusion ALK kinase using a targetedinhibitor, such as WI-131 and/or WI-154

Example 5 ALK Fusion Proteins Drive Growth and Survival of TransformedMammalian Cell Line

In order to confirm that expression of one or more of the ALK fusionproteins can transform normal cells into a cancerous phenotype, 3T#cells may be transformed with the cDNA constructs described above(Example 2), which express the EML4-ALK (short and long variants) orTFG-ALK fusion proteins, respectively.

Briefly, cells are maintained in RPMI-1640 medium (Invitrogen) with 10%fetal bovine serum (FBS) (Sigma) and 1.0 ng/ml IL-3 (R&D Systems).Production of retroviral supernatant and transduction is carried out aspreviously described. See Schwaller et al., Embo J. 17(18): 5321-33(1998). 3T3 cells are transduced with retroviral supernatant containingthe MSCV-Neo/EML4-ALK (or TFG-ALK) vector and selected for G418 (1mg/ml). The ability of transformed cells to grow on soft agar is thenaccessed by plating transduced cells after the cells are washed threetimes in PBS. If desired, for dose response curves, cells are treatedwith siRNA against ALK as described above (see Example 3), and thenumber of viable cells is determined with the CellTiter 96 AQ_(ueous)One solution cell proliferation assay (Promega). IC₅₀ may be calculatedwith the use of OriginPro 6.1 software (OriginLab). The percentage ofapoptotic cells at 48 hours may be determined by flow cytometricanalysis of Cleaved-Caspase-3 using an antibody specific for this target(Cell Signaling Technology). Such an analysis would show that theexpression of EML4-ALK fusion protein (short or long variant) or TFG-ALKfusion protein can transform the 3T3 cells and confirm survival andgrowth on soft agar when these cells are driven by the ALK fusionprotein, and further that inhibition of ALK expression in thetransformed cells leads to decreased viability and increased apoptosis.

Example 6 Detection of EML4-ALK Fusion Protein Expression in Human SolidTumors Using FISH Assay

The presence of the EML4-ALK fusion protein (short variant) in humanNSCLC tumor samples was detected using a fluorescence in situhybridization (FISH) assay, as previously described. See, e.g., Verma etal. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork, N.Y. (1988). Over 200 paraffin-embedded human NSCLC tumor sampleswere examined.

An ALK dual color, break-apart rearrangement probe was obtained fromVysis (Vysis, Dowers Grove, Ill., USA) and used according to themanufacturer's instructions with the following modifications. In brief,paraffin embedded tissue sections were re-hydrated and subjected tomicrowave antigen retrieval in 0.01M Citrate buffer (pH 6.0) for 11minutes. Sections were digested with Protease (4 mg/ml Pepsin, 2000-3000U/mg) for 25 minutes at 37° C., dehydrated and hybridized with the FISHprobe set at 37° C. for 18 hours. After washing,4′,6-diamidino-2-phenylindole (DAPI; mg/ml) in Vectashield mountingmedium (Vector Laboratories, Burlingame, Calif.) was applied for nuclearcounterstaining.

The ALK rearrangement probe contains two differently labeled probes onopposite sides of the breakpoint of the ALK gene (at nucleotide 3171) inthe wild type sequence (SEQ ID NO: 6). When hybridized, the native ALKregion will appear as an orange/green fusion signal, while rearrangementat this locus (as occurs in the EML4-ALK deletion mutants) will resultin separate orange and green signals. See FIG. 6.

The FISH analysis revealed a relatively low incidence of this shortvariant EML4-ALK mutation in the sample population studied (one out of229 samples). However, given the high incidence of NSCLC worldwide (over151,00 new cases in the U.S. annually, alone), there are expected to bea significant number of patients that harbor this mutant ALK, whichpatients may benefit from an ALK-inhibiting therapeutic regime.

Example 7 Detection of ALK Fusion Protein Expression in Human SolidTumors Using PCR Assay

The presence of one or more ALK fusion proteins in a human solid tumorsample may be also be detected using either genomic or reversetranscriptase (RT) polymerase chain reaction (PCR), previouslydescribed. See, e.g., Cools et al., N. Engl. J. Med. 348:1201-1214(2003). Briefly and by way of example, solid tumor samples may beobtained from a patient having, e.g. NSCLC, using standard techniques.PCR probes against truncated ALK kinase or EML4-ALK fusion protein(short or long variant) or TFG-ALK fusion protein are constructed.RNeasy Mini Kit (Qiagen) may be used to extract RNA from tumor samples.DNA may be extracted with the use of DNeasy Tissue Kit (Qiagen). ForRT-PCR, first-strand cDNA is synthesized from, e.g., 2.5 μg of total RNAwith the use, for example, of SuperScript™ III first-strand synthesissystem (Invitrogen) with oligo (dT)₂₀.

Then, the ALK fusion gene is amplified with the use of primer pairs,e.g. EML4-202 and ALK-GSP3 (see Example 2 above). For genomic PCR,amplification of the fusion gene may be performed with the use ofPlatinum Taq DNA polymerase high fidelity (Invitrogen) with primerpairs, e.g. EML4-202 and ALK-GSP3 (see Example 2, above). Such ananalysis will identify a patient having a solid tumor characterized byexpression of the truncated ALK kinase (and/or EML4-ALK fusionprotein(s) or TFG-ALK fusion protein), which patient is a candidate fortreatment using an ALK-inhibiting therapeutic, such as WHI-131 and/orWHI154.

In one non-limiting example, DNA (e.g., genomic or cDNA) can beamplified using real-time PCR. Real-time PCR (also called quantitativereal time PCR or QPCR) is particularly useful, for example, toquantitatively compare differences in the amount of cDNA in twodifferent sample (e.g., a control or wild-type sample and a NSCLC tumorsample). Methods for performing real-time PCR are known, including theuse of DNA-binding dyes (e.g., SYBR Green), and the use of a fluorescentreporter probe, such as a probe that hybridizes to a nucleic acidsequence encoding either ALK, EML4, or the fusion junction between oneof the EML4-ALK fusion polypeptides of the invention (or a nucleic acidsequence complementary to these sequences), that incorporates afluorescent reporter at one end and a quencher of the fluorescentreporter at the other end of the probe. The close proximity of thereporter to the quencher prevents detection of its fluorescence. In thisexample, the fluorescent reporter located at the 5′ end of the probe.The 5′ to 3′ exonuclease activity of the Taq polymerase used in the PCRbreaks breaks the reporter-quencher proximity and thus allows unquenchedemission of fluorescence, which can be detected. An increase in theproduct targeted by the fluorescent reporter probe at each PCR cycletherefore causes a proportional increase in fluorescence due to thebreakdown of the probe and release of the fluorescent reporter.

What is claimed is:
 1. A method for detecting the presence of a mutantAnaplastic Lymphoma Kinase (ALK) polypeptide in a circulating tumorcell, said method comprising the steps of: (a) obtaining a circulatingtumor cell from a patient having cancer; (b) utilizing at least onereagent that detects a mutant ALK polypeptide, to determine whether amutant ALK polypeptide is present in said circulating tumor cell.
 2. Themethod of claim 1, wherein said at least one reagent is an antibody thatbinds to a mutant ALK polypeptide.
 3. The method of claim 1, wherein thedetection in step (b) is implemented in an immunohistochemistry format.4. The method of claim 1, wherein the detection in step (b) isimplemented in a flow cytometry format.
 5. The method of claim 1,wherein said cancer is lung cancer.
 6. The method of claim 5, whereinsaid lung cancer is non-small cell lung cancer (NSCLC).